U.S. patent application number 15/929387 was filed with the patent office on 2020-09-17 for methods, systems, and compositions for determining blood clot formation, and uses thereof.
The applicant listed for this patent is The Children's Medical Center Corporation, President and Fellows of Harvard College. Invention is credited to Riccardo Barrile, Andrew L. Frelinger, III, Donald E. Ingber, Abhishek Jain, Alan David Michelson, Andries D. van der Meer.
Application Number | 20200292531 15/929387 |
Document ID | / |
Family ID | 1000004869724 |
Filed Date | 2020-09-17 |
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United States Patent
Application |
20200292531 |
Kind Code |
A1 |
Ingber; Donald E. ; et
al. |
September 17, 2020 |
Methods, Systems, And Compositions For Determining Blood Clot
Formation, And Uses Thereof
Abstract
A method is directed to determining a thrombosis function and
includes flowing a fluid sample over a surface having a fixed
endothelial cell monolayer. The method further includes stimulating
the fixed endothelial cell monolayer to induce formation of a clot,
the clot being formed via interaction between the fixed endothelial
cell monolayer and the fluid sample. In response to the clot
formation, the method further includes determining a thrombosis
function associated with the fluid sample and the fixed endothelial
cell monolayer.
Inventors: |
Ingber; Donald E.; (Boston,
MA) ; Jain; Abhishek; (Roslindale, MA) ; van
der Meer; Andries D.; (Enschede, NL) ; Michelson;
Alan David; (Boston, MA) ; Frelinger, III; Andrew
L.; (North Reading, MA) ; Barrile; Riccardo;
(Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College
The Children's Medical Center Corporation |
Cambridge
Boston |
MA
MA |
US
US |
|
|
Family ID: |
1000004869724 |
Appl. No.: |
15/929387 |
Filed: |
April 29, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15576235 |
Nov 21, 2017 |
10732172 |
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PCT/US2016/033686 |
May 22, 2016 |
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15929387 |
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62310166 |
Mar 18, 2016 |
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62165272 |
May 22, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/5027 20130101;
G01N 33/86 20130101; G01N 2800/226 20130101; G01N 33/5064
20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; B01L 3/00 20060101 B01L003/00; G01N 33/86 20060101
G01N033/86 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was made with Government Support under
N66001-11-1-4180 awarded by the Space and Naval Warfare Systems
Center of the U.S. Department of Defense, and under
HR0011-13-C-0025 awarded by the Defense Advanced Research Projects
Agency of the U.S. Department of Defense. The government has
certain rights in the invention.
Claims
1-65. (canceled)
66. A method of making microfluidic devices, comprising: a)
providing a microfluidic device comprising a channel; b) culturing
endothelial cells in the channel so as to form a lumen; c) fixing
the endothelial cells; and d) shipping the device.
67. The method of claim 66, wherein the fixing is done with a
fixative agent selected from the group consisting of formaldehyde,
paraformaldehyde, formalin, glutaraldehyde, mercuric chloride-based
fixatives, Helly's solution, Zeneker's solution, precipitating
fixatives, ethanol, methanol, acetone, dimethyl suberimidate,
Bouin's fixative, a decellularization solvent, a detergent, a high
pH solution, and a combination of two or more thereof.
68. The method of claim 66, wherein the method further comprises
the step of storing the microfluidic device after step c).
69. The method of claim 68, wherein the microfluidic device is
stored at a temperature between 4.degree. C. and 10.degree. C.
70. The method of claim 68, wherein the microfluidic device is
stored at a temperature of about 4.degree. C. or lower.
71. The method of claim 68, wherein the microfluidic device is
stored at room temperature.
72. The method of claim 68, wherein the microfluidic device is
stored for 1 day or longer.
73. The method of claim 68, wherein the microfluidic device is
stored for 5 days or longer.
74. A method of making microfluidic devices, comprising: a)
providing a microfluidic device comprising a channel; b) culturing
endothelial cells in the channel of each microfluidic device so as
to form a lumen; c) fixing the endothelial cells; and d) storing
the plurality of microfluidic devices.
75. The method of claim 74, wherein the fixing is done with a
fixative agent selected from the group consisting of formaldehyde,
paraformaldehyde, formalin, glutaraldehyde, mercuric chloride-based
fixatives, Helly's solution, Zeneker's solution, precipitating
fixatives, ethanol, methanol, acetone, dimethyl suberimidate,
Bouin's fixative, a decellularization solvent, a detergent, a high
pH solution, and a combination of two or more thereof.
76. The method of claim 74, wherein the microfluidic device is
stored at a temperature between 4.degree. C. and 10.degree. C.
77. The method of claim 74, wherein the microfluidic device is
stored at a temperature of about 4.degree. C. or lower.
78. The method of claim 74, wherein the microfluidic device is
stored at room temperature.
79. The method of claim 74, wherein the microfluidic device is
stored for 1 day or longer.
80. The method of claim 74, wherein the microfluidic device is
stored for 5 days or longer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of U.S.
Provisional Patent Application Ser. No. 62/165,272, filed on May
22, 2015, and U.S. Provisional Patent Application Ser. No.
62/310,166, filed on Mar. 18, 2016, each of which is hereby
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to quantifying a
thrombosis-related function in vitro based on physiologically
relevant conditions, and, more particularly, to a microfluidic
system having fluid flow interaction between a fixed endothelial
layer and cells (such as platelets) in a fluid sample.
BACKGROUND OF THE INVENTION
[0004] Generally, the vascular endothelium and shear stress are
critical determinants of hemostasis and platelet function in vivo,
and yet, current diagnostic and monitoring devices do not fully
incorporate endothelial function under flow in their assessment.
Therefore, current diagnostic and monitoring devices can be
unreliable and inaccurate. Furthermore, it is challenging to
include the endothelium in assays for clinical laboratories or
point-of-care settings because living cell cultures are not
sufficiently robust.
[0005] More specifically, mutual signaling between endothelium and
activated platelets is widely recognized as critical for regulation
of hemostasis and thrombotic disorders associated with various
diseases, including atherosclerosis, sepsis, and diabetes. Yet, no
practical diagnostic assays exist that can measure cross-talk
between platelets and inflamed vessel walls in the presence of
physiological shear. Over the last decade or so, multiple flow
chambers and microfluidic devices that contain microchannels have
been lined by living endothelium and exposed to flowing blood to
study the basic science of thrombosis. While these devices have
been very useful in advancing research, they have not been used in
clinical settings due to the difficulty in maintaining living
endothelial cells in them. Specifically, because it is extremely
difficult to maintain the viability of living cell cultures for
extended times in non-controlled settings, it is virtually
impossible to rely on these assays. Therefore, the only
microfluidic devices that are currently being deployed in clinical
diagnostic settings are lined with collagen to mimic thrombus
formation and platelet aggregation induced in response to vascular
injury, and, thus, they fail to capture the physiological interplay
between endothelial cells, platelets and fluid shear stress that is
so relevant to hemostasis in inflammatory diseases.
[0006] Additionally, pulmonary microvascular thrombosis is a
catastrophic condition amounting to a large number of patient
deaths worldwide. Despite significant progress in understanding
fundamental biology of lung hemostasis and thrombosis, it is still
very difficult to predict response and study mechanism of action of
potential drug candidates to humans. This is partly so because
currently available in vitro assays do not recapitulate
physiologically-relevant forces, such as shear stress, and animal
models can be very complex allowing limited experimental
manipulation, making it impossible to dissect and study
intercellular signaling.
[0007] More specifically, pulmonary intravascular thrombosis and
platelet activation initiating from, for example, acute lung injury
("ALI"), acute chest syndrome ("ACS"), pulmonary hypertension
("PH"), chronic obstructive pulmonary disease ("COPD"), and acute
respiratory distress syndrome ("ARDS"), are causes of significantly
high patient mortality and morbidity. Therefore, pulmonary
intravascular thrombosis and platelet activation are also promising
and emerging therapeutic targets to save and prolong patient life.
Although epithelial injury, endothelial dysfunction, and in situ
thrombotic lesions are observed often in human patients in chronic
pulmonary diseases, animal models of pulmonary dysfunction are
still unable to completely mimic the altered hemostasis and
hemodynamic complexity of the lung. Importantly, animal models can
be very complex and it may be impossible to study cell-cell
interactions between multiple tissues independently of each other
during blood clotting or drug administration. Based on this type of
limitations, along with ethical barriers associated with animal
models, it is desirable to advance in vitro disease models of
pulmonary thrombosis that can mimic human organ-level functionality
and complement or reduce reliance on animal studies, to enable more
reliable basic research and make drug discovery more efficient.
[0008] In vitro, commercially available coagulation and platelet
function technologies also have serious limitations due to the fact
that they do not incorporate physiological tissue-tissue or
cell-cell interactions, and relevant fluid dynamics of blood cells,
which are key determinants of thrombosis. In research laboratories,
dishes and transwell plates have been used for decades to culture
cells and study basic biology, but these are static systems, highly
non-physiological and cannot recapitulate tissue or organ-level
functionality. For example, this type of systems cannot
recapitulate blood flow or breathing of a lung.
[0009] To incorporate blood perfusion, parallel plate-flow chambers
have been widely applied in the past three decades or so to measure
thrombus formation and platelet adhesion kinetics. However, being
macroscale devices, these chambers do not mimic small blood
vessels, typically do not incorporate endothelium, and require
large blood sample volumes for analysis.
[0010] More recently, microfluidic devices lined with human
endothelial cells have shown that endothelial activation, platelet
adhesion and fibrin formation in the presence of physiological
shear can be somewhat visualized. However, these devices are also
limited in studying organ-level pulmonary thrombosis, in part
because they do not include the role of live epithelial cells,
dynamic platelet-endothelial interactions (e.g., activation,
aggregation, adhesion, translocation, and embolization) in the
lumen that occur over large spatiotemporal scales, and often do not
incorporate perfusion of whole blood.
[0011] Recently, microfluidic technology has been advanced to
demonstrate an organ-level in vitro model of a lung and pulmonary
edema, where alveolar epithelial and endothelial cells were
co-cultured in two overlaying chambers, respectively. Fibrin
formation in the alveolar chamber was analyzed in the presence of
an inflammatory cytokine IL-2 and in the presence of flow and
relevant cyclic stretch. However, this type of lung-on-a-chip model
still lacks relevant functionality for mimicking relevant
foundational conditions of pulmonary thrombosis. For example, the
endothelial chamber only contains one side cultured with the cells
and hence, it does not contain an endothelial lumen. Based on this
limitation, the device is not appropriate for perfusing whole blood
and for studying blood cell-endothelial interactions. In fact,
other than a dilute suspension of neutrophils, none of the blood
cells or platelets has been perfused or analyzed in this type of
device, in its physiological concentration.
[0012] Another limitation of the long-on-a-chip model is that it
uses non-primary epithelial cell lines, A549 or NCI-H441. Although
this type of model mimics certain aspects of human lung function,
it is not ideal in the context of mimicking
physiologically-relevant hemostasis and thrombosis, as they are
derived from tumors and, therefore, can potentially alter
endothelial and platelet function.
[0013] Therefore, there is a continuing need for solving the above
and other problems.
SUMMARY OF THE INVENTION
[0014] According to one aspect of the present invention, a
microfluidic device is lined with a human endothelium that is
chemically fixed, but still retains its ability to modulate
hemostasis under continuous flow in vitro. For example, according
to one method, microfluidic channels are seeded with collagen and
endothelial cells and are left either untreated or treated with
tumor necrosis factor-.alpha. (TNF-.alpha.). The cells are, then,
fixed with formaldehyde. Recalcified citrated whole blood (0.5 mL)
from healthy volunteers or patients taking antiplatelet medication
is perfused and platelet coverage is recorded. The chemopreserved
endothelialized device is lined with a bioinspired material that
supports formation of platelet-rich thrombi in the presence of
physiological shear, similar to a living arterial vessel.
Furthermore, the method demonstrates the potential clinical value
of the chemopreserved endothelialized device by showing that
thrombus formation and platelet function are measurable within
minutes using a small volume of whole blood taken from subjects
receiving antiplatelet medications. The method further demonstrates
potentially greater reliability than standard platelet function
tests and collagen-coated perfusion chambers.
[0015] According to another aspect of the present invention, a
microengineered lung-on-chip device is used for studying human
pulmonary blood clotting and platelet-endothelial interaction
dynamics. The lung-on-chip is a microfluidic device populated with
primary alveolar cells ("AE") localized within a top channel and
vascular endothelial cells in a bottom compartment. The top channel
and the bottom compartment are separated by a matrix-coated
membrane. Whole blood is perfused in the vascular compartment while
the epithelium is stimulated with a cytokine or endotoxin, and
platelet-endothelial interactions are recorded in real-time. To
quantify the dynamics of the platelet-endothelial interactions, a
stochastic analytical method is provided that is highly sensitive
to changes in endothelial and platelet activation. In vitro, the
presence of alveolar epithelium is shown to be beneficial for
reconstituting pulmonary thrombosis in response to an inflammatory
stimulus of lipopolysaccharide ("LPS"). Additionally, this model is
used in drug development by analyzing the effect of a novel
protease activator receptor-1 ("PAR1") antithrombotic compound,
termed parmodulin 2 ("PM2"), and demonstrate that PM2 has an
endothelial cytoprotective effect in response to LPS-mediated
inflammation. The lung-on-chip device reconstitutes organ-level
functionality that accurately reflects many aspects of human
pulmonary thrombosis and appears to offer a valuable platform for
drug development.
[0016] According to one aspect of the present invention, a method
is directed to determining a thrombosis function and includes
flowing a fluid sample over a surface having a fixed endothelial
cell monolayer. The method further includes stimulating the fixed
endothelial cell monolayer to induce formation of a clot, the clot
being formed via interaction between the fixed endothelial cell
monolayer and the fluid sample. In response to the clot formation,
the method further includes determining a thrombosis function
associated with the fluid sample and the fixed endothelial cell
monolayer.
[0017] According to another aspect of the invention, a microfluidic
system is directed to determining a thrombosis function. The
microfluidic system includes a compartment having a surface with a
fixed endothelial cell monolayer, the compartment being configured
to receive a fluid sample flowing over the surface such that cells
in the fluid sample interact with the fixed endothelial cell
monolayer. The microfluidic system further includes a detection
module configured to detect interaction between the cells and the
fixed endothelial cell monolayer, and to determine a function of
the cells in the fluid sample.
[0018] According to yet another aspect of the invention, a device
is directed to simulating a function of a tissue. The device
includes a first structure defining a first microchannel and
configured to have a fluid sample flowing within, the fluid sample
including platelets. The device further includes a second structure
defining a second microchannel, and a membrane located at an
interface region between the first microchannel and the second
microchannel. The membrane has a first side facing toward the first
microchannel and a second side facing toward the second
microchannel, the membrane separating the first microchannel from
the second microchannel. The first side of the membrane includes a
fixed endothelial cell monolayer, the second side of the membrane
including at least one layer of tissue-specific cells. The device
further includes a detection module configured to detect
interaction between the platelets and the fixed endothelial cell
monolayer. The detection module is further configured to determine
a function of the platelets in the fluid sample.
[0019] According to yet another aspect of the invention, a system
is directed to quantifying thrombosis in vitro based on
physiological conditions. The system includes a solid substrate
having a surface with a fixed endothelial cell monolayer, and a
detection module configured to receive the solid substrate. The
detection module is further configured to detect spatial and
temporal interaction between cells in a fluid sample and the
surface of the solid substrate when the fluid sample is flowed over
the surface along a flow axis. The system further includes one or
more controllers configured to store time-lapse data of detectable
signals collected from the detection module, wherein the detectable
signals represent spatial and temporal interaction between the
cells and the surface.
[0020] The one or more controllers are further configured to
generate a kymograph from at least a portion of the stored
time-lapse data, wherein a time axis of the kymograph indicates at
least a portion of the time-lapse duration, a space axis of the
kymograph indicating the detectable signals along the flow axis.
The one or more controllers are further, yet, configured to
determine, based on the generated kymograph, a rate of fluctuation
in a coefficient of variation (CV) of the detectable signals to
generate a temporal cell dynamics index, and to determine either
(i) the presence of reactive cells in the fluid sample when the
temporal cell dynamics index is higher than a temporal control
value, or (ii) the absence of reactive cells in the fluid sample
when the temporal cell dynamics index is no more than the temporal
control value. The system further includes a display module for
displaying content that is based in part on output determined by
the one or more controllers, wherein the content includes a signal
indicative of either presence or absence of at least one of
reactive cells or cell aggregation in the fluid sample.
[0021] According to yet another aspect of the invention, a method
is directed to quantifying thrombosis in vitro based on
physiological conditions. The method includes providing a solid
substrate having a surface with a fixed endothelial cell monolayer,
and detecting, via a detection module, spatial and temporal
interaction between cells in a fluid sample and the surface of the
solid substrate when the fluid sample is flowed over the surface
along a flow axis. The method further includes storing, via one or
more controllers, time-lapse data of detectable signals that are
collected from the detection module, the detectable signals
representing spatial and temporal interaction between the cells and
the surface. The method also includes generating a kymograph, via
at least one of the one or more controllers, from at least a
portion of the stored time-lapse data, a time axis of the kymograph
indicating at least a portion of the time-lapse duration, a space
axis of the kymograph indicating the detectable signals along the
flow axis.
[0022] Based on the generated kymograph, the method determines, via
at least one of the more controllers, a rate of fluctuation in a
coefficient of variation (CVO) of the detectable signals to
generate a temporal cell dynamics index. The method further
includes determining, via at least one of the one or more
controllers, (i) the presence of reactive cells in the fluid sample
when the temporal cell dynamics index is higher than a temporal
control value, or (ii) the absence of reactive cells in the fluid
sample when the temporal cell dynamics index is no more than the
temporal control value. The method further includes displaying, via
a display module, content that is based in part on output
determined by the one or more controllers, the content including a
signal indicative of either presence or absence of at least one
reactive cells or cell aggregation in the fluid sample.
[0023] According to yet another aspect of the invention, a system
is directed to determining dynamics of platelets in a fluid sample.
The system includes a solid substrate having a surface with a fixed
endothelial cell monolayer, and a detection module configured to
receive the solid substrate. The detection module is further
configured to detect spatial and temporal interaction between cells
in a fluid sample and the surface of the solid substrate when the
fluid sample is flowed over the surface along a flow axis. The
system further includes one or more controllers configured to store
time-lapse data of detectable signals collected from the detection
module, wherein the detectable signals represent spatial and
temporal interaction between the cells and the surface.
[0024] The one or more controllers are further configured to
generate a kymograph from at least a portion of the stored
time-lapse data, wherein a time axis of the kymograph indicates at
least a portion of the time-lapse duration, a space axis of the
kymograph indicating the detectable signals along the flow axis.
The one or more controllers are also configured to determine, based
on the generated kymograph, a rate of fluctuation in a coefficient
of variation (CV) of the detectable signals to generate a platelet
dynamics index, the platelet dynamics index being one or more of a
temporal platelet dynamics index and a spatial platelet dynamics
index. The one or more controllers are further configured to
determine either (i) the presence of reactive platelets in the
fluid sample when the platelet dynamics index is higher than a
control value, or (ii) the absence of reactive platelets in the
fluid sample when the platelet dynamics index is no more than the
control value. The system further includes a display module for
displaying content that is based in part on output determined by
the one or more controllers, wherein the content includes a signal
indicative of either presence or absence of at least one of
reactive platelets or platelet aggregation in the fluid sample.
[0025] In addition, the inventors have shown that the fixed
endothelial cell monolayers that have been stored for a period of
time (e.g., at least about 5 days or more) without freezing were
still applicable for platelet function analysis. Not only can this
concept be applied to platelet function analysis, but it can also
be generally extended to analyses of interaction dynamics of other
cell types.
[0026] Further, instead of merely determining area-averaged
platelet adhesion--a static analysis--as regularly used in existing
platelet function assessment, the inventors have developed novel
analytical methods to quantify temporal and/or spatial changes in
the way of how cells interact with each other and/or to a surface.
In some embodiments, the inventors have showed that the resulting
characteristic temporal and spatial indices were sensitive enough
to distinguish activated platelets (e.g., due to inflamed
endothelial cells) and non-activated platelets. Thus, the temporal
and spatial indices can be used as markers to diagnose diseases or
disorders (e.g., platelet-associated disease or disorder), to
select appropriate therapy (e.g., anti-platelet and/or
anti-inflammation therapy), to monitor treatment efficacy (e.g., to
prevent recurrent thrombosis or bleeding), drug screening and/or to
determine drug toxicology. Accordingly, embodiments of various
aspects described herein relate to methods, systems, and
compositions for determining dynamic interaction of cells with each
other, and/or with other cell types, and uses thereof.
[0027] One aspect described herein relates to a method of
determining cell function. The method comprises (a) flowing a fluid
sample over a surface comprising a monolayer of cells of a first
type thereon; and (b) detecting interaction between cells of a
second type in the fluid sample and the monolayer of cells of the
first type. The function of the cells of the second type in the
fluid sample can then be determined based on the detected cell
interaction.
[0028] In some embodiments, the fixed monolayer of cells of the
first type can comprise endothelial cells, and the cells of the
second type in the fluid sample can comprise blood cells, e.g.,
platelets. Accordingly, another aspect provided herein relates to a
method of determining platelet function, which comprises (a)
flowing a fluid sample over a surface comprising a fixed
endothelial cell monolayer thereon; and (b) detecting interaction
between blood cells (e.g., platelets) in the fluid sample and the
fixed endothelial cell monolayer.
[0029] In some embodiments, the fixed cell monolayer (e.g., fixed
endothelial cell monolayer) can be derived from fixing target cell
extract (e.g., endothelial cell extract) and/or target
cell-associated proteins (e.g., endothelial cell-associated
proteins) that are adhered to the surface. The target
cell-associated proteins can comprise proteins secreted by the
target cells and/or present on the target cell surface. Where the
target cell-associated proteins comprise endothelial
cell-associated proteins, examples of endothelial cell-associated
proteins can include, but are not limited to, any art-recognized
procoagulatory and/or anti-coagulatory proteins. In some
embodiments, the endothelial cell-associated proteins can comprise
von Willebrand factor and/or tissue factor (TF).
[0030] Any cell-comprising fluid sample can be flowed over the
fixed cell monolayer and it can vary depending on what target cells
to be analyzed. In some embodiments, the fluid sample can comprise
a blood sample, a serum sample, a plasma sample, a lipid solution,
a nutrient medium, or a combination of two or more thereof. In some
embodiments when the fluid sample comprises a blood sample, the
method can further comprise removing red blood cells from the blood
sample prior to flowing the blood sample over the surface. In some
embodiments, the fluid sample flowing over the surface in the
methods described herein can comprise calcium ions and/or magnesium
ions.
[0031] The surface over which the fluid sample flows can be a
surface of any fluid-flowing conduit disposed in a solid substrate
that is compatible to the fluid sample and the cells. In some
embodiments, the solid substrate can comprise a cell culture
chamber. For example, in one embodiment, the surface can be a wall
surface of a microchannel. In one embodiment, the surface can be a
surface of a membrane.
[0032] In some embodiments where the surface is a surface of a
membrane, the membrane can be configured to separate a first
chamber (e.g., a first microchannel) and a second chamber (e.g., a
second microchannel) in a microfluidic device.
[0033] In some embodiments, the microfluidic device can be
configured to comprise an organ-on-chip device. An exemplary
organ-on-chip can comprise a first chamber (e.g., a first
microchannel), a second chamber (e.g., a second microchannel), and
a membrane separating the first chamber and the second chamber. In
these embodiments, a first surface of the membrane facing the first
chamber can comprise the fixed cell monolayer (e.g., fixed
endothelial cell monolayer) thereon, and a second surface of the
membrane facing the second chamber can comprise tissue-specific
cells adhered thereon. In some embodiments, the membrane can be
replaced or embedded with extracellular matrix proteins (e.g., but
not limited to collagen, laminin, etc.). In some embodiments, the
membrane can also comprise smooth muscle cells and/or
fibroblasts.
[0034] In some embodiments, the fixed cell monolayer (e.g., fixed
endothelial cell monolayer) can be derived from fixing a layer of
cells of the first type (e.g., an endothelial cell monolayer) that
has been grown on the surface for a period of time. For example,
the layer of cells of the first type (e.g., an endothelial cell
monolayer) can grow on the surface until it reaches confluence and
is then subjected to a fixation treatment as described herein.
[0035] Various methods for fixing cells that are adhered to a
surface are known in the art and can be used herein to generate a
fixed cell monolayer. In some embodiments, the cell monolayer
(e.g., endothelial cell monolayer) can be physically fixed by
drying and/or dehydration. In some embodiments, the cell monolayer
(e.g., endothelial cell monolayer) can be physically fixed by
exposing to air, and/or washing with alcohol, acetone or a solvent
that removes water and/or lipids. In some embodiments, the cell
monolayer (e.g., endothelial cell monolayer) can be fixed with a
chemical fixative. Non-limiting examples of chemical fixatives
include formaldehyde, paraformaldehyde, formalin, glutaraldehyde,
mercuric chloride-based fixatives (e.g., Helly and Zenker's
solution), precipitating fixatives (e.g., ethanol, methanol, and
acetone), dimethyl suberimidate (DMS), Bouin's fixative, and a
combination of two or more thereof. In one embodiment, the chemical
fixative for fixing the cell monolayer (e.g., endothelial cell
monolayer) can comprise paraformaldehyde. In some embodiments, the
cell monolayer (e.g., endothelial cell monolayer) can be fixed with
a decellularization solvent that stabilizes surface membrane
protein configuration and cytoskeleton of a cell. For example, the
decellularization solvent can comprise an aqueous solution
comprising a detergent and/or a high pH solution.
[0036] The fixed cell monolayer (e.g., fixed endothelial cell
monolayer) can be derived from a cell line or cells collected from
a subject. In some embodiments, cells collected from a subject can
be reprogrammed to form pluripotent stem cells, which are then
differentiated into target cells to generate a fixed cell
monolayer.
[0037] The fixed cell monolayer (e.g., fixed endothelial cell
monolayer) can be derived from cells of any condition. In some
embodiments, the fixed cell monolayer (e.g., fixed endothelial cell
monolayer) can be derived from healthy cells. In some embodiments,
the fixed cell monolayer (e.g., fixed endothelial cell monolayer)
can be derived from diseased cells. In some embodiments, the
diseased cells can be derived from a subject (e.g., a healthy
subject or a subject diagnosed with a disease or disorder of
interest). In some embodiments, the diseased cells can be generated
by contacting healthy cells (e.g., healthy endothelial cells) with
a condition-inducing agent (e.g., inflammation-inducing agent)
prior to the fixation treatment. The condition-inducing agent
(e.g., inflammation-inducing agent) can comprise a physical
stimulus, a chemical agent, a biological agent, a molecular agent,
or a combination of two or more thereof.
[0038] By detecting interaction between cells (e.g., blood cells
such as platelets) in the fluid sample and the fixed cell monolayer
(e.g., fixed endothelial cell monolayer), temporal and/or spatial
dynamics of the cells in the fluid sample interacting with each
other and/or to the fixed cell monolayer can be measured. In some
embodiments, the measured temporal and/or spatial dynamics of cell
interaction measured can comprise cell adhesion, cell detachment,
cell translocation, and cell embolization/aggregation. In some
embodiments, the measured temporal and/or spatial dynamics of cell
interaction can comprise binding dynamics of the cells (e.g., blood
cells such as platelets) to the fixed cell monolayer (e.g., fixed
endothelial cell monolayer), binding dynamics of the cells (e.g.,
blood cells such as platelets) to each other, or a combination
thereof.
[0039] Depending on cell detection methods, the cells in the fluid
sample can be label-free or labeled, e.g., with a detectable label.
An exemplary detectable label can comprise a fluorescent label.
[0040] Any art-recognized cell detection methods can be used to
detect interaction between the cells in the fluid sample and the
fixed cell monolayer. In some embodiments, an imaging-based method
can be used. An exemplary imaging-based method can comprise
time-lapse microscopy.
[0041] The inventors have showed that the fixed endothelial cell
monolayer can be stored for a period of time without undermining
its applicability to platelet dynamics analysis. Accordingly, in
some embodiments, the surface comprising the fixed cell monolayer
(e.g., fixed endothelial cell monolayer) can have been stored for a
period of time prior to flowing the fluid sample over the surface.
In some embodiments, the fixed cell monolayer (e.g., fixed
endothelial cell monolayer) can be stored at a non-freezing
temperature. For example, in some embodiments, the fixed cell
monolayer (e.g., fixed endothelial cell monolayer) can be stored at
room temperature. In some embodiments, the fixed cell monolayer
(e.g., fixed endothelial cell monolayer) can be stored at a
temperature of about 4.degree. C. or lower. In some embodiments,
the fixed cell monolayer (e.g., fixed endothelial cell monolayer)
can be stored at a temperature of about 4.degree. C.-10.degree.
C.
[0042] The period of time to store the fixed cell monolayer (e.g.,
fixed endothelial cell monolayer) can vary with the selected
storage temperature. In some embodiments, the period of time can be
at least about 1 day or longer. In some embodiments, the period of
time can be at least about 5 days or longer.
[0043] The fluid sample can be flowed over the surface comprising
the fixed cell monolayer (e.g., fixed endothelial cell monolayer)
at a pre-determined shear rate or flow rate. For example, the fluid
sample can be flowed over the surface at a flow rate that generates
a physiological or pathological wall shear rate. For example, the
physiological or pathological wall shear rate can range from about
50 sec.sup.-1 to about 10,000 sec.sup.-1.
[0044] The fixed cell monolayer (e.g., fixed endothelial cell
monolayer) and the fluid sample can be derived from the same
subject or from different sources.
[0045] In some embodiments, the fixed cell monolayer can comprise a
fixed endothelial cell monolayer, and the fluid sample cells can
comprise blood cells such as platelets. Accordingly, in these
embodiments, the system can be used to determine spatial dynamics
of blood cells such as platelets in a fluid sample.
[0046] The methods and/or systems described herein can provide
tools to diagnose a disease or disorder induced by cell dysfunction
or abnormal cell-cell interaction in a subject. Accordingly,
another aspect described herein relates to a method of determining
if a subject is at risk, or has, a disease or disorder induced by
cell dysfunction or abnormal cell-cell interaction. The method
comprises: (a) flowing a fluid sample of the subject over a surface
comprising a fixed cell monolayer thereon; (b) detecting
interaction of cells in the fluid sample between each other and/or
with the fixed cell monolayer; and (d) identifying the subject to
be at risk, or have the disease or disorder induced by cell
dysfunction when the cell-cell interaction is higher than a
control; or identifying the subject to be less likely to have a
disease or disorder induced by cell dysfunction when the cell-cell
interaction is no more than the control.
[0047] In some embodiments, the living or fixed cell monolayer used
in the methods described herein can be subject-specific.
[0048] In some embodiments, the method of determining if a subject
is at risk, or has a disease or disorder induced by cell
dysfunction and/or abnormal cell-cell interaction can be used for
diagnosis and/or prognosis of a disease or disorder induced by
blood cell dysfunction (e.g., platelet dysfunction), and/or guiding
and/or monitoring of an anti-platelet and/or anti-inflammation
therapy. Accordingly, in some embodiments, the fixed endothelial
cell monolayer can comprise a fixed endothelial cell monolayer. The
fixed endothelial cell monolayer can be subject-specific. In some
embodiments, the fluid sample can comprise blood cells such as
platelets. Thus, a method of determining if a subject is at risk,
or has a disease or disorder induced by blood cell dysfunction
(e.g., platelet dysfunction) is also described herein. Non-limiting
examples of the disease or disorder induced by blood cell
dysfunction (e.g., platelet dysfunction) include, but are not
limited to thrombosis, an inflammatory vascular disease (e.g.,
sepsis, or rheumatoid arthritis), a cardiovascular disorder (e.g.,
acute coronary syndromes, stroke, or diabetes mellitus),
vasculopathies (e.g., malaria, disseminated intravascular
coagulation), or a combination of two or more thereof.
[0049] Compositions for determining cell-cell interaction are also
described herein. In one aspect, the composition comprises (a) a
solid substrate having a surface comprising a fixed monolayer of
cells of a first type thereon; and (b) a fluid sample in contact
with the surface, wherein the fluid sample comprises cells of a
second type.
[0050] In some embodiments, the fixed monolayer of cells of the
first type can comprise a fixed endothelial cell monolayer. In some
embodiments, the cells of the second type in the fluid sample can
comprise blood cells such as platelets.
[0051] In some embodiments, the fluid sample can comprise a blood
sample.
[0052] The fixed cell monolayer can comprise fixed cells (e.g.,
fixed endothelial cells), fixed cell extract(s) (e.g., fixed
endothelial cell extract(s)), and/or fixed cell-associated proteins
(e.g., fixed endothelial cell-associated proteins) that are adhered
to the surface.
[0053] In some embodiments, the fixed cell monolayer (e.g., fixed
endothelial cell monolayer) can be derived from fixing a cell layer
(e.g., an endothelial cell monolayer) that has been grown on the
surface for a period of time, e.g., until the cell layer reaches
confluence.
[0054] The surface with which the fluid sample is in contact can be
a surface of any fluid-flowing conduit disposed in a solid
substrate. The solid substrate can be any solid substrate that is
compatible to the fluid sample and the fixed cell monolayer.
Non-limiting examples of the solid substrate include a cell culture
device, a microscopic slide, a cell culture dish, a microfluidic
device, a microwell, and any combinations thereof.
[0055] In one embodiment, the surface can be a wall surface of a
microchannel. In one embodiment, the surface can be a surface of a
membrane. In some embodiments where the surface is a surface of a
membrane, the membrane can be configured to separate a first
chamber (e.g., a first microchannel) and a second chamber (e.g., a
second microchannel) in a microfluidic device.
[0056] In some embodiments, the microfluidic device can be
configured to comprise an organ-on-chip device. An exemplary
organ-on-chip can comprise a first chamber (e.g., a first
microchannel), a second chamber (e.g., a second microchannel), and
a membrane separating the first chamber and the second chamber. In
these embodiments, a first surface of the membrane facing the first
chamber can comprise the fixed cell monolayer (e.g., fixed
endothelial cell monolayer) thereon, and a second surface of the
membrane facing the second chamber can comprise tissue-specific
cells adhered thereon. In some embodiments, the membrane can be
replaced or embedded with extracellular matrix proteins (e.g., but
not limited to collagen, laminin, etc.). In some embodiments, the
membrane can also comprise smooth muscle cells and/or
fibroblasts.
[0057] For example, in some embodiments, the methods, systems,
and/or compositions described herein can be configured to permit a
blood cell-comprising fluid sample (e.g., platelet-comprising fluid
sample) flowing over a more reliable and physiologically relevant
endothelialized surface inflamed by a cytokine, thus mimicking the
in vivo endothelium-blood cell (e.g., platelet) crosstalk
environment, e.g., in a normal or diseased state. The blood cell
(e.g., platelet) dynamics (e.g., adhesion, translocation and/or
detachment) can be recorded and quantified, which is not possible
with the existing gold standard tests. As the blood cell (e.g.,
platelet) function/interaction can be reproduced even when the live
endothelial cells are fixed, the compositions with a fixed
endothelial cell monolayer described herein can be stored under
standard laboratory conditions for a period of time (e.g., days or
weeks) and still remain functional. Thus, the compositions
described herein can be operated near patients' bedside, e.g., in
clinics or hospitals, to determine blood cell (e.g., platelet)
dysfunction, e.g., for diagnosis of a disease or disorder induced
by blood cell (e.g., platelet) dysfunction.
[0058] In some embodiments, the compositions described herein can
further comprise tissue-specific cells. For example, in some
embodiments, a microfluidic device can comprise a first chamber
(e.g., a first microchannel), a second chamber (e.g., a second
microchannel), and a membrane separating the first chamber and the
second chamber, wherein a first surface of the membrane facing the
first chamber can comprise a fixed endothelial cell monolayer
thereon, and a second surface of the membrane facing the second
chamber can comprise tissue-specific cells adhered thereon. A fluid
comprising blood cells (e.g., blood or blood substitute) can be
introduced into the first chamber such that blood cells can
interact with the fixed endothelial cell monolayer. In some
embodiments, the fixed endothelial monolayer can be an inflamed or
diseased endothelial cell monolayer. By incorporating luminal blood
cell fluid transport (e.g., a fluid comprising blood cells such as
platelets) over a fixed endothelial cell monolayer and live culture
of tissue specific cells, a physiologically relevant in vitro model
of blood cell-induced inflammation can be created to probe its
pathophysiology and/or to permit drug screening.
[0059] Additional aspects of the invention will be apparent to
those of ordinary skill in the art in view of the detailed
description of various embodiments, which is made with reference to
the drawings, a brief description of which is provided below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] FIG. 1 is a perspective illustration of a microfluidic
device.
[0061] FIG. 2 is top view illustration of a single microfluidic
chip with a plurality of microfluidic devices.
[0062] FIG. 3 is a fluorescence micrograph of a microchannel
covered with human umbilical vein endothelial cells ("HUVECs").
[0063] FIG. 4A is a confocal immunofluorescence microscopic image
showing a top view of a microchannel section with HUVECs.
[0064] FIG. 4B shows a front view of the microchannel section of
FIG. 4A.
[0065] FIG. 4C shows a side view of the microchannel section of
FIG. 4A.
[0066] FIG. 5 is a graph that shows fluorescence measured after
immunostaining a fixed endothelium with ICAM-1.
[0067] FIG. 6 is a graph that shows fluorescence measured after
immunostaining a fixed endothelium with VCAM-1.
[0068] FIG. 7 is a graph that shows fluorescence measured after
immunostaining a fixed endothelium with VWF.
[0069] FIG. 8 is a graph that shows fluorescence measured after
immunostaining a fixed endothelium with a tissue factor.
[0070] FIG. 9 is a plurality of representative maximum intensity
projection micrographs with fluorescently labeled platelets
adhering to a chemopreserved endothelium.
[0071] FIG. 10 is a graph that shows platelet coverage when blood
is perfused inside a microchannel that is lined with a living or
fixed endothelium.
[0072] FIG. 11A is a fluorescent micrograph showing fibrin that is
formed along with platelet aggregates on a fixed endothelium (scale
bar--200 .mu.m).
[0073] FIG. 11B is a fluorescent micrograph showing fibrin that is
formed along with platelet aggregates on a fixed endothelium (scale
bar--20 .mu.m).
[0074] FIG. 12 is a graph illustrating platelet coverage on a fixed
endothelium that is pretreated with TNF-.alpha. when blood samples
are perfused through a microfluidic device.
[0075] FIG. 13 is a graph illustrating light transmission
aggregometry of blood samples containing different doses of
abciximab using either ADP or collagen as an agonist.
[0076] FIG. 14 is a graph illustrating platelet coverage when blood
samples containing different doses of the drug abciximab are
perfused through collage-coated microfluidic devices.
[0077] FIG. 15 is a graph illustrating platelet coverage on a fixed
endothelium that has been pretreated with TNF-.alpha. when blood
samples from healthy donors are perfused through microfluidic
devices.
[0078] FIG. 16 is a graph illustrating light transmission
aggregometry of healthy versus antiplatelet treated blood samples
using ADP or collagen as an agonist.
[0079] FIG. 17 is a graph illustrating platelet coverage when
healthy versus subject blood samples are perfused through
collagen-coated microfluidic devices.
[0080] FIG. 18 is an illustration of a microfluidic blood vessel
with cultured endothelial cells.
[0081] FIG. 19 shows fluorescence micrographs depicting a section
of an imaged microchannel showing platelet accumulation (left to
right) on collagen, a healthy blood vessel, and a TNF-.alpha.
stimulated vessel.
[0082] FIG. 20 shows fluorescence micrographs depicting a section
of an imaged microchannel with platelet accumulation after 4
minutes of laser-induced injury on a mouse cremaster arteriole
(scale bar--.mu.m 25).
[0083] FIG. 21 shows fluorescent micrographs of a large section of
a vascular chamber with intravascular thrombus formation in
collagen (top image), and TNF-.alpha. stimulated endothelium in a
dose dependent manner (bottom three images).
[0084] FIG. 22 is a graph illustrating ICAM-1 expression on the
endothelial cells after stimulation with TNF-.alpha..
[0085] FIG. 23 is a graph illustrating a sensitivity analysis of a
platelet endothelial dynamics algorithm.
[0086] FIG. 24 is a conceptual schematic of a human lung showing
alveoli interacting with neighboring blood vessels during
hemostasis or pulmonary dysfunction.
[0087] FIG. 25 is a perspective view illustration of a microfluidic
device with two compartments separated by a thin porous
membrane.
[0088] FIG. 26 is a side view illustration of the microfluidic
device of FIG. 25.
[0089] FIG. 27 shows visual stacks of confocal micrographs with
junctional structures, after twelve days of co-culture.
[0090] FIG. 28 is a chart showing vascular ICAM-1 measured after
TNF-.alpha. stimulation relative to untreated cells in the presence
of alveolar epithelial cells (AE).
[0091] FIG. 29 is a chart showing platelet-endothelial dynamics in
a microfluidic device that follows a similar trend as ICAM-1 of
FIG. 28.
[0092] FIG. 30 shows fluorescent micrographs with platelets (left),
fibrin (middle), and merged (right) on an endothelial surface when
stimulated by TNF-.alpha..
[0093] FIG. 31 is a chart showing vascular ICAM-1 that is measured
after LPS stimulation relative to untreated cells in the presence
or absence of alveolar epithelial cells (AE).
[0094] FIG. 32 is a chart showing platelet-endothelial dynamics
measured in a microfluidic device, in the presence or absence of
the alveolar epithelial cells (AE).
[0095] FIG. 33 shows fluorescence micrographs with platelet
aggregates and fibrin at the end of blood perfusion through a
microfluidic device.
[0096] FIG. 34 is a chart showing barrier permeability measured
after LPS stimulation, relative to untreated cells in the presence
or absence of the alveolar epithelial cells (AE).
[0097] FIG. 35 shows representative confocal micrographs with gap
junctions under no treatment or LPS treatment, in the presence of a
blood vessel alone or with epithelium (AE).
[0098] FIG. 36 shows fluorescent micrographs illustrating evolution
of blood clots (left to right) in a cremaster artery of the
mouse.
[0099] FIG. 37 is a chart showing platelet-endothelial dynamics
computed on fluorescent time-series of platelets.
[0100] FIG. 38 is an illustration showing a microfluidic device
that contains alveolar epithelial cells (AE) treated with LPS and a
vessel treated with parmodulin (PM2).
[0101] FIG. 39 is a chart showing platelet-endothelial dynamics
that are measured in a microfluidic device containing AE cells.
[0102] FIG. 40 is a chart illustrating platelet coverage on a
microchip covered with collagen.
[0103] FIG. 41 shows a coefficient of variation (CV) colormap of a
single thrombus formed in a laser injured mouse in vivo.
[0104] FIG. 42 shows histological sections representing sections of
a mouse lung with clots.
[0105] FIG. 43 shows a representative kymograph of a small section
of a channel with an attachment and detachment pattern of platelets
on a collagen surface.
[0106] FIG. 44 shows a representative kymograph of a small section
of a channel with an attachment and detachment pattern of platelets
on a vessel that is untreated.
[0107] FIG. 45 shows a representative kymograph of a small section
of a channel with an attachment and detachment pattern of platelets
on a vessel that is TNF-.alpha. treated.
[0108] FIG. 46 shows a chart shows a coefficient of variance (CV)
of a fluorescence signal observed over time at a representative
single pixel location of an image time-series of platelet
accumulation, as plotted in the kymographs shown in FIGS.
43-45.
[0109] FIG. 47 shows a top image representative of a coefficient of
variation (CV) colormap of a large section of a vessel, and a
bottom image with a graph showing the CV across the length of the
channel at a representative width location for a collagen-treated
vessel.
[0110] FIG. 48 shows a top image representative of a coefficient of
variation (CV) colormap of a large section of a vessel, and a
bottom image with a graph showing the CV across the length of the
channel at a representative width location for an untreated
vessel.
[0111] FIG. 49 shows a top image representative of a coefficient of
variation (CV) colormap of a large section of a vessel, and a
bottom image with a graph showing the CV across the length of the
channel at a representative width location for vessel treated with
TNF-.alpha..
[0112] FIG. 50 shows a graph illustrating the interpercentile range
(95.sup.th-5.sup.th percentile value) of the coefficient of
variation (CV) plotted in the graphs illustrated in FIGS. 47-49, as
a measure of depicting spatial heterogeneity in platelet
accumulation.
[0113] FIG. 51 illustrates an exemplary organ-on-chip (OOC) device
in accordance with one embodiment of the present disclosure.
[0114] FIG. 52 is a cross-section of the organ-on-chip (OOC) device
taken along line 52-52 of FIG. 51, illustrating first and second
microchannels of the organ-on-chip (OOC) device.
[0115] FIG. 53 is a cross-section of the organ-on-chip (OOC) device
taken along line 53-53 of FIG. 52, illustrating fluid flow between
the first microchannel and the second microchannel of the
organ-on-chip (OOC) device of FIG. 51.
[0116] FIGS. 54A-54E are images and schematic diagrams of a
biomimetic platelet function analyzer (.mu.PFA) according to one or
more embodiments described herein. (FIG. 54A) Schematic of the
multilayered microfluidic device comprising a first channel and a
second channel, wherein the first channel and the second channel
are separated by a permeable membrane. The side of the membrane
facing the first channel can comprise an endothelium adhered
thereto. The other side of the membrane facing the second channel
can comprise astrocytes or other cell types of interest. Shear
stresses and/or fluid flow (e.g., whole blood or blood flow) can be
induced in the first channel. The multilayered microfluidic device
can optionally comprise a vacuum channel on one or both sides of
the first and the second channel. (FIG. 54B) Picture of one
embodiment of a device showing blood passing through it when pulled
by a syringe pump. (FIG. 54C) Schematic drawing (top view) of the
vascular chamber showing inlet, outlet and optional pressure ports.
(FIG. 54D) A fluorescent micrograph of endothelial cells (green/top
channel, CD31 staining) and astrocytes (red/bottom channel/GFAP
staining) co-cultured in one embodiment of the device described
herein. Such device can then be perfused with blood. (FIG. 54E)
(left panels) A sectional view of the vascular chamber coated with
HUVECs and inflamed with tumor necrosis factor (TNF-.alpha.).
(right panels) The endothelial ICAM-1 expression is increased with
increase in TNF concentration.
[0117] FIG. 55 is a set of data showing platelet adhesion on
different surfaces. (Left panel) Bar graph showing area-averaged
platelet adhesion rate on collagen, unstimulated endothelium and
cytokine-stimulated endothelium. (Right panels) Snapshots of a
section of the vascular chamber after 15 minutes of whole blood
flow containing labeled platelets. Scale bar=50 .mu.m. **
P<0.001
[0118] FIG. 56 is a set of data showing temporal dynamics of
platelets interacting with different surfaces. (Left panel) Bar
graph showing Temporal Platelet Dynamics (TPD) indices varying with
different surfaces, namely, collagen, unstimulated endothelium and
cytokine-stimulated endothelium. (Right panels) Kymographs of a
section of the vascular chamber perfused with whole blood
containing labeled platelets. Scale bar=50 .mu.m (vertical
direction). ** P<0.001
[0119] FIG. 57 is a set of data showing spatial dynamics of
platelets interacting with different surfaces. (Left panel) Bar
graph showing Spatial Platelet Dynamics (SPD) indices varying with
different surfaces, namely, collagen, unstimulated endothelium and
cytokine-stimulated endothelium. (Right panels) Time-averaged
coefficient of variation (CV) maps of a section of the vascular
chamber perfused with whole blood containing labeled platelets.
Scale bar=50 .mu.m. ** P<0.001
[0120] FIG. 58 is a set of micrographs showing formaldehyde-fixed
human umbilical vein endothelial cells (HUVECs) in the device
according to one or more embodiments described herein. (Left panel)
von Willebrand factor staining (green). (Right panel) Tissue factor
(TF) staining (green).
[0121] FIGS. 59A-59C are bar graphs showing area averaged platelet
adhesion (FIG. 59A), Temporal Platelet Dynamics (TPD) (FIG. 59B)
and Spatial Platelet Dynamics (SPD) (FIG. 59C) of platelets over
collagen (COL*) and endothelium fixed for 1 day or 5 days (ENDO*).
** P<0.001
[0122] FIG. 60 is a set of confocal images showing a cross-section
of a two-compartment organ-on-a-chip with co-cultures of HUVECs
(top compartment) and human astrocytes (bottom compartment). The
HUVECs and astrocytes were both treated overnight with 100 ng/ml
TNF-.alpha.. The endothelial compartment (in which the endothelial
cells were cultured on all walls of a channel) was perfused with
whole blood for about 15 minutes. (Left panel) Platelets (red) were
observed to be mainly on the walls of the endothelial compartment,
while fibrin has formed mostly in the static (no shear) astrocyte
compartment due to the reaction between blood fibrinogen and
thrombin. The fibrin passed through the endothelial compartment
(high shear). (Right panel) F-actin/nuclear staining shows
astrocyte localizations on the membrane and the floor of the bottom
compartment.
[0123] FIG. 61 is a block diagram showing an exemplary system for
use in the methods described herein, e.g., for determining temporal
and/or spatial dynamics of cells (e.g., platelets) binding to each
other and/or a cell monolayer (e.g., an endothelial cell
monolayer).
[0124] FIG. 62 is a block diagram showing an exemplary system for
use in the methods described herein, e.g., for determining temporal
and/or spatial dynamics of cells (e.g., platelets) binding to each
other and/or a cell monolayer (e.g., an endothelial cell
monolayer).
[0125] FIG. 63 is an exemplary set of instructions on a computer
readable storage medium for use with the systems described herein
to determine temporal dynamics of cells (e.g., platelets) binding
to each other and/or a cell monolayer (e.g., an endothelial cell
monolayer).
[0126] FIG. 64 is an exemplary set of instructions on a computer
readable storage medium for use with the systems described herein
to determine spatial dynamics of cells (e.g., platelets) binding to
each other and/or a cell monolayer (e.g., an endothelial cell
monolayer). In some embodiments, the exemplary set of instructions
can further comprise a portion of the instructions from FIG. 63 to
compute temporal dynamics of the cells (e.g., platelets) binding to
each other and/or a cell monolayer. When both Aggregation (spatial)
Index (FIG. 64) and Embolization (temporal) Index (FIG. 63) are
used to determine cell dynamic behavior, to diagnose disease,
and/or to monitor therapy, in some embodiments, both indices can be
greater than their respective control values. In some embodiments,
both indices can be lower than their respective control values. In
some embodiments, one index can be greater than its respective
control value, while another index can be lower than its respective
control value. For example, samples from patients with
hypercoagulable disorders can show normal/strong platelet
aggregation (e.g., represented by a high Aggregation Index), but
very low "embolization."
[0127] FIGS. 65A-65C depict an embodiment of a platelet dynamics
assessment device as described herein. FIG. 65A depicts a schematic
of the microfluidic device for quantifying platelet dynamics on a
living endothelium under flow when cultured within a hollow
microchannel (400 .mu.m wide, 100 .mu.m high, 2 cm long). Human
whole blood is stored in a reservoir at the inlet (left) and pulled
by a syringe pump attached to the outlet (right) at a flow rate of
30 .mu.l/min (shear rate: 750 sec.sup.-1). Fluorescently tagged
platelets that interact with the endothelium are visualized over
time within a central region of the long section of the channel
using automated microscopy. FIG. 65B depicts a photograph of the
microfluidic platelet assessment chip containing 6-channels (bar,
15 mm). FIG. 65C depicts a representative fluorescence micrograph
of platelet-rich thrombi that form on the TNF-.alpha. treated
endothelial surface in this device when whole blood is perfused.
The thrombi contain both platelets (red) and fibrin (green) (bar,
left: 100 .mu.m, right: 25 .mu.m). A 3-dimensional confocal
reconstruction of platelet-rich thrombi formed on the
endothelium-lined microfluidic channel, stimulated by cytokine
TNF-.alpha. can be generated.
[0128] FIGS. 66A-66B depict the comparison of platelet aggregation
on collagen versus living endothelium. FIG. 66A depicts the image
acquisition and analysis protocol according to one embodiment
described herein. Fluorescent micrographs were acquired every 30
sec for a total of 15 min; 10 (1.times.10) image tiles were
captured at each time step and stitched together to form a
panoramic view, resulting in an image time series (K). FIG. 66B
depicts fluorescence micrographs of the microchannel when coated
with collagen (COL; top) or lined with endothelium (HUVEC; bottom)
are shown on the left. Representative image tiles of platelets
interacting with the collagen-coated surface (top) or the surface
of endothelium stimulated with different doses of TNF-.quadrature.
(bottom) 10 min after initiating blood flow are shown at the right
(bar, 200 .mu.m).
[0129] FIGS. 67A-67B depict the quantitative analysis of platelet
adhesion and thrombus formation using an Aggregation Index (AI).
FIG. 67A depicts representative coefficient of variance (CV) maps,
produced using the "fire" color map, showing platelet adhesion
patterns on a collagen surface (COL) versus endothelial (HUVEC)
lined surface stimulated with different doses of TNF.alpha.. Color
bar indicates the intensity of aggregation/thrombi (white is
greatest; blood flow was from left to right; bar, 200 .mu.m). FIG.
67B depicts a graph showing platelet aggregation indices (AI)
derived from maps shown in FIG. 67A. The time series stack (K) is
projected across time computing the temporal coefficient of
variance (CV) at each spatial pixel (M), and the AI is the
inter-quartile range (IQR) of M. Note that the unstimulated
endothelium does not induce platelet adhesion or thrombus
formation, whereas the amount and variability of the aggregation
pattern increases in TNF.alpha. dose-dependent manner on stimulated
endothelial cells; this results in a rise of AI with increasing
TNF.alpha. dose (0 ng TNF.alpha./ml; 5 ng TNF.alpha./ml; 100 ng
TNF.alpha./ml; n=3,**p<0.01).
[0130] FIGS. 68A-68B depict the quantitative analysis of
translocation and embolization of platelet-rich thrombi using an
Embolization Index (EI). FIG. 68A depict representative
size-adjusted kymographs showing embolization pattern of platelets
on a collagen surface (COL) or endothelium (HUVEC) stimulated with
different TNF.alpha. doses. FIG. 68B depicts a graph showing
platelet Embolization Indices (EI) derived from kymographs shown in
FIG. 68A. The time series stack (K) was averaged across the width
of the channel and a kymograph (a temporal map of platelet
dynamics) was generated (N); the EI is the CV of N. Note that,
platelets remained adherent to the collagen surface over time and
did not translocate, resulting in a low EI. The unstimulated
endothelium did not induce translocation and/or embolization of
platelet-rich thrombi whereas these dynamical processes increased
in a TNF.alpha. dose-dependent manner (0 ng TNF.alpha./ml; 5 ng
TNF.alpha./ml; 100 ng TNF.alpha./ml; n=3,**p<0.01).
[0131] FIGS. 69A-69E depict whole blood platelet analysis on
chemically preserved endothelium in a microchannel according to one
embodiment described herein. FIG. 69A is a schematic diagram
depicting platelet thrombus formation over a natural versus
chemopreserved endothelium. In a microchannel covered on all sides
with an untreated living endothelium, whole blood flows without
clotting (left). In contrast, platelet-rich thrombus forms if the
endothelium is prestimulated by a proinflammatory cytokine, such as
TNF-.alpha., due to expression of procoagulatory proteins at its
surface (right). The responses of blood under flow shown in the
figures at the left can be reconstituted using similar
microchannels that are lined by a chemically preserved endothelium.
FIG. 69B depicts a schematic of one embodiment of the microdevice
described herein. The inlet is a blood reservoir (dia. 3.5 mm) and
it is pulled via a syringe pump at the outlet (dia. 1.5 mm)
connected to tubing (not shown). The dotted region (.about.2.5
mm.times.500 .mu.m) is visualized over time using automated
fluorescence microscopy. FIG. 69C depicts endothelial engineering
on the microchip. Confocal immunofluorescence microscopic images
show a section of the microchannel containing adherent HUVECs when
viewed from above (left) and in side view (right). The dotted
region represents the analyzed area of platelet accumulation
(green, VE cadherin; blue, nuclear DAPI; bar, 200 .mu.m). FIG. 69D
depicts quantitative analysis of platelet adhesion and aggregation
on living vs. chemopreserved endothelial substrate. (Left) The
platelet coverage on both living (white bar) and fixed (shaded bar)
endothelium increases in a TNF-.alpha. dose-dependent manner (n=4).
No significant difference in platelet coverage was observed using
living versus chemopreserved endothelium. (Right) Representative
maximum intensity projection micrographs showing fluorescently
labeled platelet-rich thrombi adhering to the chemopreserved
endothelium in a TNF-.alpha. dose-dependent manner. The statistical
analysis was performed using 2-way ANOVA (Sidak's multiple
comparison test). (bar, 200 .mu.m). FIG. 69E depicts Tissue Factor
(TF, blue) and von Willebrand Factor (vWF, purple) expression on
untreated (white bar) vs. stimulated (shaded bar) chemopreserved
endothelial substrate. Error bars, standard error of mean (s.e.m.).
* P<0.05 in all graphs.
[0132] FIGS. 70A-70F are bar graphs depicting analysis of the
chemopreserved endothelium covered microchip to monitor
antiplatelet therapy. FIG. 70A shows that the platelet coverage
over the TNF-.alpha. stimulated chemopreserved endothelium
decreases with increase in abciximab drug concentration (n=4). The
statistical analysis was performed using 1-way ANOVA (Sidak's
multiple comparison test). FIG. 70B shows that when blood is
perfused at a shear of 750 sec.sup.-1 on a collagen microfluidic
device, there is an insignificant decrease in platelet adhesion and
aggregation with increase in abciximab dosage (n=3) whereas, (as
shown in FIG. 70C) platelets aggregate in the presence of ADP
(white bar) and collagen (shaded bar) as agonists, only when
control blood is used. In the presence of drug, no aggregation is
observed on the light transmission aggregometry (n=5). (For FIGS.
70B and 70C, N.S=non-significant; statistical analysis based on
1-way ANOVA (Sidak's multiple comparison test)). FIG. 70D shows
that an untreated chemopreserved endothelium (white bar) is
quiescent for both healthy donors and patients who are on chronic
use of aspirin alone or both aspirin and clopidogrel, but in
comparison to healthy donors, patients result in significantly
lower platelet coverage on the TNF-.alpha. stimulated
chemopreserved endothelium (shaded bar)(n=11). The statistical
analysis was performed using one-way ANOVA (Sidak's multiple
comparison test). FIG. 70E shows that blood from subjects who are
on antiplatelet therapy, showed insignificant difference in
aggregation compared to healthy controls using a collagen coated
microfluidic device (n=11, p=0.4493--non-significant based on
unpaired t-test results (two-tailed)). FIG. 70F shows that blood
from subjects who are on antiplatelet therapy, exhibited
significantly less aggregation compared to healthy controls using a
light transmission aggregometry with ADP (white bar) and collagen
(shaded bar) as agonists. (n=11, *: P<0.05 based on 2-way ANOVA
analysis (Sidak's multiple comparison test)). * P<0.05 in all
graphs. N.S.=non-significant.
[0133] FIGS. 71A-71B depict the engineering of one embodiment of a
responsive endothelium-lined microfluidic channel described herein.
FIG. 71A depicts confocal immunofluorescence microscopic images
showing the entire length of the microchannel containing adherent
human umbilical cord endothelial cells (HUVECs) shown when viewed
from above (Top) and in cross-sectional views (Bottom) (green, VE
cadherin; blue, nuclear DAPI; bar, 300 .mu.m). To generate a
3-dimensional confocal reconstruction of endothelium-lined
microfluidic channel, sequential images obtained along the
microchannel containing adherent human umbilical cord endothelial
cells (HUVECs) were acquired using Leica SP5.times.MP inverted
confocal microscope. The virtual volume was processed using Huygens
deconvolution software and rendered with Imaris (Green, VE
cadherin; blue, nuclear DAPI). FIG. 71B depicts a graph (left) and
immunofluorescence microscopic views of the cultured endothelium
stained for intercellular adhesion molecule-1 (ICAM-1) at left or
F-actin at right, showing dose-dependent activation of ICAM-1 (left
images) when stimulated with tumor necrosis factor alpha
(TNF-.alpha.)(green, ICAM-1 or F-actin; blue, DAPI; bar, 300
.mu.m).
[0134] FIG. 72 is a set of bar graphs showing expression of tissue
factor (TF) and von Willebrand factor (vWF), respectively, on
TNF-.alpha. inflamed endothelium. HUVECs were cultured on
PDMS-coated 24 well plates for 48 h and left untreated or
stimulated with 5 or 100 ng/ml TNF-.alpha.. The effect of
TNF-.alpha. on TF (left panel) and vWF (right panel) expression on
the endothelial cell surface was estimated by measuring
immunofluorescence intensity, normalized with respect to the
untreated case. (*P<0.05, n=3)
[0135] FIGS. 73A and 73B are data graphs showing area averaged
platelet adhesion rate. FIG. 73A depicts area averaged platelet
adhesion rate on a surface calculated using automated Otsu image
thresholding algorithm. The percentage area covered was calculated
as the ratio of number of pixels with the value of unity to the
size of the binary image and plotted against time. The dotted line
is the linear regression curve fit and the adhesion rate is the
slope of the regression line. FIG. 73B depicts area-averaged
platelet adhesion rate. n=3,**p<0.01
[0136] FIG. 74 is a set of data graphs showing platelet adhesion
and aggregation dynamics. Graphical representation of the
coefficient of variance (CV) image M(x,y). On a collagen (COL) and
healthy endothelial (HUVEC 0 ng/ml) surface, the range of variance
is narrow. However, the platelet patterns on TNF-.alpha. treated
endothelium (HUVEC) are heterogeneous and fluctuate in a
dose-dependent manner. The inter-quartile range (IQR) of the signal
is termed platelet aggregation index (AI).
[0137] FIG. 75 shows an exemplary image acquisition and analysis
protocol for platelet coverage. Platelets were visualized using
time-lapse fluorescence imaging (LD Plan Neofluar 20.times., NA
0.4; Zeiss Axio Observer; Hamamatsu ORCA C11440 CMOS digital
camera) using an exposure time of 200 ms. Images were tiled to
create a composite panoramic view (18,600 pixels long and 2,050
pixels wide; 1 pixel=0.325 .mu.m). In step 1, a timeseries (K) of a
10-frame panorama (6 mm long.times.0.665 mm wide region of the
microchannel), at a lapse of every 30 seconds was recorded. Images
were archived as OME-TIFF format files, and image analysis was
performed using Zeiss Zen 2012 imaging software and MATLAB 2014
routines. The resulting image stack was maximum intensity projected
along time (step 2), thresholded, segmented (step 3) and cropped to
the central 200 .mu.m of the channel width (step 4) for analysis.
Finally, platelet coverage was computed from the binary image as
the ratio of bright pixels (intensity value=1) to the total number
of pixels in the image (step 5).
[0138] FIG. 76 is a set of fluorescent images showing platelet-rich
thrombus formation in the microfluidic device after blood
perfusion. Fluorescent micrograph shows fibrin (green) is formed
along with platelet aggregates (red) in collagen and TNF-.alpha. (5
ng/ml) stimulated chemopreserved endothelium after recalcified
citrated whole blood is perfused through the device. The platelet
aggregates are small and more uniformly distributed over the
collagen compared to the inflamed endothelial surface. (bar, 100
.mu.m)
[0139] While the invention is susceptible to various modifications
and alternative forms, specific embodiments have been shown by way
of example in the drawings and will be described in detail herein.
It should be understood, however, that the invention is not
intended to be limited to the particular forms disclosed. Rather,
the invention is to cover all modifications, equivalents, and
alternatives falling within the spirit and scope of the invention
as defined by the appended claims.
DETAILED DESCRIPTION
[0140] While this invention is susceptible of embodiment in many
different forms, there is shown in the drawings and will herein be
described in detail preferred embodiments of the invention with the
understanding that the present disclosure is to be considered as an
exemplification of the principles of the invention and is not
intended to limit the broad aspect of the invention to the
embodiments illustrated.
[0141] As used herein, the term "monolayer" refers to a single
layer of cells on a growth surface, on which no more than 10%
(e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%) of the cells
are growing on top of one another, and at least about 90% or more
(e.g., at least about 95%, at least 98%, at least 99%, and up to
100%) of the cells are growing on the same growth surface. In some
embodiments, all of the cells are growing side-by side, and can be
touching each other on the same growth surface. The condition of
the cell monolayer can be assessed by any methods known in the art,
e.g., microscopy, and/or immunostaining for cell-cell adhesion
markers. In some embodiments where the fixed cell monolayer
comprises a fixed endothelial cell monolayer, the condition of the
endothelial cell monolayer can be assessed by staining for any
art-recognized cell-cell adhesion markers in endothelial cells,
e.g., but not limited to VE-cadherin.
[0142] In some embodiments, the monolayer can be substantially
confluent. As used herein, the term "confluent" generally indicates
that the cells have formed a coherent monocellular layer on a
growth surface, so that virtually all the available growth surface
is used. As used herein, the term "substantially confluent"
indicates that most of the cells growing on the same surface are in
contact with each other around the cell periphery, and interstices
can remain, such that over about 70% or more, including, e.g., over
about 80%, over about 90%, over about 95% or more, and up to 100%,
of the available growth surface is used. The term "available growth
surface" as used herein refers to sufficient surface area to
accommodate a cell. Thus, small interstices between adjacent cells
that cannot accommodate an additional cell do not constitute
"available growth surface." Accordingly, in some embodiments, the
fixed cell monolayer (e.g., fixed endothelial cell monolayer) can
be derived from fixing a layer of cells of the first type (e.g., an
endothelial cell monolayer) that has been grown on the surface for
a period of time. In some embodiments, the period of time can vary
with degree of confluence, cell proliferation rate, number of
initially seeded cells, number of cell culture passages, or a
combination thereof. For example, the layer of cells of the first
type (e.g., an endothelial cell monolayer) can grow on the surface
until it is substantially confluent and is then subjected to a
fixation treatment as described herein. In some embodiments where
the layer of cells of the first type comprises endothelial cells,
the endothelial cell culture can be cultured for at least about 24
hours or longer to reach an intact and confluent monolayer.
[0143] In some embodiments, the monolayer can be exposed to or
stimulated by an agent, e.g., a condition-inducing agent, prior to
fixation. In these embodiments, the cells in the monolayer can
overgrow and form plexiform lesions, prior to fixation.
[0144] The fixed cell monolayer (e.g., fixed endothelial cell
monolayer) can be derived from a cell line (e.g., primary cell
lines), stem cells, or cells collected from a subject. In some
embodiments, target cells to be cultured on the surface can be
collected from a subject. For example, to form a fixed endothelial
cell monolayer, endothelial cells can be collected from a subject.
In some embodiments, endothelial cells can be derived from stem
cells or induced pluripotent stem cells. For example, skin
fibroblasts can be collected from a subject and reprogrammed to
form pluripotent stem cells, which are then differentiated into
subject-specific target cells (e.g., endothelial cells) to form a
fixed cell monolayer (e.g., a fixed endothelial cell monolayer).
Methods to derive different types of differentiated cells from
induced pluripotent stem cells are known in the art. For example,
vascular endothelium can be derived from induced pluripotent stem
cells using the methods as described, e.g., in Adams et al., Stem
Cell Reports (2013) 1:105-113.
[0145] The fixed cell monolayer (e.g., fixed endothelial cell
monolayer) can be derived from cells of any condition or state
(e.g., but not limited to wild-type, healthy state, mutant,
disease-specific, and stimulated state). In some embodiments, the
fixed cell monolayer (e.g., fixed endothelial cell monolayer) can
be derived from healthy cells or wild-type cells. As used herein,
the term "healthy" refers to a state without any symptoms of any
diseases or disorders, or not identified with any diseases or
disorders, or not on any physical, chemical and/or biological
treatment, or a state that is identified as healthy by skilled
practitioners based on microscopic examinations. As used herein,
the term "wild-type" refers to a natural state without any genetic
manipulation.
[0146] In some embodiments, the fixed cell monolayer (e.g., fixed
endothelial cell monolayer) can be derived from disease-specific
cells. As used herein, the term "disease-specific" refers to a
state of cells that recapitulates at least one characteristic
associated with a disease, disorder or an injury, or different
stages thereof. In some embodiments, the term "disease-specific"
can refer to a specific stage or grade of a disease, disorder or an
injury. Examples of diseases, disorders, or injuries can be related
to any organ or tissue, e.g., but not limited to, blood vessel,
lung, brain, nerve network, blood-brain-barrier, vascular, kidney,
liver, heart, spleen, pancreas, ovary, testis, prostate, skin, eye,
ear, skeletal muscle, colon, intestine, and esophagus. In some
embodiments where the fixed cell monolayer comprises a fixed
endothelial cell monolayer, the endothelial cells can recapitulate
at least one characteristic associated with a vascular and/or
inflammatory disease or disorder.
[0147] The disease-specific cells can be either obtained from a
biopsy of a patient carrying the disease, disorder or an injury, or
inducing a healthy cell with a condition-inducing agent that is
known to induce the cell to acquire at least one characteristic
associated with the disease, disorder, or injury, prior to a
fixation treatment. For example, a condition-inducing agent can
comprise an environmental or physical agent such as radiation; a
chemical or biological agent, e.g., but not limited to, cytokines
described herein and/or pathogens; a molecular agent (e.g., but not
limited to a pathogen-derived toxin such as lipopolysaccharides
(LPS), and/or a candidate drug/compound that is known to cause
endothelial activation or thrombotic toxicity), or a combination of
two or more thereof.
[0148] In some embodiments, the fixed cell monolayer (e.g., fixed
endothelial cell monolayer) can be derived from stimulated cells.
As used herein, the term "stimulated" refers to a state of cells
that are responsive to a condition-inducing agent exposed to them.
As used herein, the term "condition-inducing agent" refers to any
agent that can cause a cell to display a phenotype that is deviated
from a basal state (without exposure to the condition-inducing
agent). The condition-inducing agent can provoke a beneficial or
adverse effect such as cytotoxic effect on the cells. Examples of a
condition-inducing agent can include, but are not limited to,
environmental or physical agents such as radiation (e.g., gamma
radiation) and mechanical stress (e.g., fluid shear stress);
proteins, peptides, nucleic acids, antigens, cytokines, growth
factors, toxins, cells (including prokaryotic and eukaryotic cells
such as virus, bacteria, fungus, parasites, and mammalian cells),
particulates (e.g., smoke particles or asbestos), particles (e.g.,
nanoparticles or microparticles, magnetic particles), small
molecules, biologics, and any combinations thereof.
[0149] In some embodiments where the fixed cell monolayer comprises
a fixed endothelial cell monolayer, the condition-inducing agent
added to the endothelial cell monolayer prior to fixation can
comprise an inflammation-inducing agent that induces endothelium
inflammation and/or activation. The inflammation-inducing agent can
comprise one or a combination of two or more of the following:
physical conditions (e.g., lack of oxygen, and disturbed flow
patterns), chemical (e.g., glucose levels, environmental
pollutants), biochemical (e.g., inflammatory molecules such as
interleukins, interferons, TNF-superfamily molecules), biological,
human cell derived (complex mixtures), or biological, non-human
cell derived (e.g., bacteria, or factors secreted by bacteria). In
some embodiments, the inflammation-inducing agent can comprise at
least one or more (e.g., at least two or more) proinflammatory
cytokines such as IL-6, IL-2, IL-10, sCD40L, interleukins and
interferons, which can be applied to produce endothelial activation
and inflammation that are involved in platelet function, activation
and aggregation. Other factors such as lipopolysaccharide (LPS),
toxins (Shiga toxin etc.), bacteria, viruses, nanoparticles,
antibodies and drug candidates can also be used as
inflammation-inducing agents to stimulate the endothelium. In some
embodiments, the inflammation-inducing agent can comprise glucose.
For example, the surface comprising the fixed endothelial cell
monolayer can be configured to provide highly fluctuating levels of
glucose to stimulate a "diabetic" surface. In some embodiments,
complex mixtures of soluble factors from tumor cells, and/or
activated white blood cells can be used as the
inflammation-inducing agents to activate the endothelium. In some
embodiments, the inflammation-inducing agents can comprise
temporary co-culture with tumor cells and/or white blood cells to
induce endothelial activation.
[0150] As used herein, the term "fixed," "fixation," or "fixing"
means that cell-associated components or materials, including,
e.g., but not limited to whole cells, cell fragments, intracellular
proteins, extracellular proteins (e.g., secreted proteins, cell
surface receptors), nucleic acid molecules, and/or cytoskeleton,
are treated with a fixative agent or composition, resulting in at
least a partial stabilization or preservation of their molecular
position, histological structure, and/or molecular function. Upon
fixation, whole cells are not alive anymore but proteins and/or
nucleic acid of the cells and cell-associated proteins and/or
nucleic acid present in the cell monolayer remain stable and
functional (e.g., ability to induce a response in other live
cells). Thus, cell fixation can provide spatial heterogeneity and
expressions of proteins and/or nucleic acid molecules that would be
expected in live culture and/or in vivo. In some embodiments, a
fixation agent or composition can result in at least about 50% or
more (including, e.g., at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 95%, at least
about 98%, or more or up to 100%) stabilization or preservation of
the cell-associated components' or materials' molecular position,
histological structure, and/or molecular function.
[0151] Various methods for fixing cells that are adhered to a
surface are known in the art and can be used herein to generate a
fixed cell monolayer. In some embodiments, cell fixation methods
can be selected to keep whole cell intact. For example, in some
embodiments, the cell monolayer (e.g., endothelial cell monolayer)
can be physically fixed by drying and/or dehydration. For example,
in some embodiments, the cell monolayer (e.g., endothelial cell
monolayer) can be physically fixed by exposing the cell monolayer
to air, and/or washing the cell monolayer with a drying solvent,
e.g., alcohol (e.g., ethanol) and acetone, or a solvent that
removes water and/or lipids. In some embodiments, the cell
monolayer (e.g., endothelial cell monolayer) can be fixed with a
chemical fixative. Non-limiting examples of chemical fixatives
include formaldehyde, paraformaldehyde, formalin, glutaraldehyde,
mercuric chloride-based fixatives (e.g., Helly and Zenker's
solution), precipitating fixatives (e.g., ethanol, methanol, and
acetone), dimethyl suberimidate (DMS), Bouin's fixative, and a
combination of two or more thereof. In one embodiment, the chemical
fixative for fixing the cell monolayer (e.g., endothelial cell
monolayer) can comprise paraformaldehyde. Accordingly, in some
embodiments, the fixed cell monolayer (e.g., endothelial cell
monolayer) can be derived by fixing whole cells adhered on a
surface.
[0152] In some embodiments, the cell layer (e.g., endothelial cell
monolayer) can be fixed with a fixative agent or composition
comprising paraformaldehyde.
[0153] Additionally or alternatively, cell fixation methods can be
selected to remove a portion of cell components and fix the
remaining cell components. For example, in some embodiments, the
cell monolayer (e.g., endothelial cell monolayer) can be fixed with
a decellularization solvent. The decellularization solvent is a
solvent that partially or completely removes or extracts cell
membranes and/or soluble components but stabilizes molecular
configuration, molecular function, and molecular position of
surface membrane proteins (e.g., membrane protein receptors),
insoluble components, and/or cytoskeleton of a cell. Methods for
fixing cells by membrane removal or extraction are known in the
art, e.g., as described in Ben-Ze'ev et al., Cell (1979) 17:
859-865; Pourati et al., Am J Physiol. (1998) 274: C1283-1289; Sims
et al., J Cell Sci. (1992) 103: 1215-1222; and Fey et al., J Cell
Biol. (1984) 98: 1973-1984. F or example, in some embodiments, the
decellularization solvent can comprise an aqueous solution
comprising a detergent (e.g., polysorbate surfactants such as Tween
20, and/or a nonionic surfactant such as Triton X-100; glycosides
such as saponins) and/or a high pH solution (e.g., an alkaline
solution such as ammonium hydroxide). Accordingly, in some
embodiments, the fixed cell monolayer can be derived from fixing
cell extract and/or cell-associated proteins that are adhered to
the surface.
[0154] In some embodiments, an additive can be added to a fixative
agent or composition to render cells permeable to ligands which
bind to intracellular moieties. Binding of ligands (e.g., but not
limited to, antibodies and detectable labels) to intracellular
moieties can be desired for purposes of visualizing, detecting, or
isolating the cells after they have been preserved. Additives that
increase the permeability of cell membrane and/or nuclear envelopes
are known in the art. For example, the organic solvent acetone,
methanol, and/or ethanol can increase the permeability of cell
membrane when preservation of protein and/or nucleic acid moieties
is/are desired.
[0155] In some embodiments, an additive can be added to a fixative
agent or composition to keep cells isosmotic which helps
preservation. For example, sugars such as sucrose and/or buffered
solutions can be added to keep cells isosmotic.
[0156] A skilled artisan can optimize the concentrations of a
fixative agent or composition added to the cell monolayer (e.g., an
endothelial cell monolayer). Typical concentrations of the fixative
agent or composition can range from about 1% (v/v) to about 10%
(v/v), and they can vary with the strength of the selected fixative
agent or composition. For example, when paraformaldehyde is used to
fix the cell monolayer (e.g., endothelial cell monolayer), the
concentration of the paraformaldehyde can range from about 1% (v/v)
to about 8% (v/v). In one embodiment, the cell monolayer (e.g.,
endothelial cell monolayer) can be fixed with paraformaldehyde at a
concentration of about 4%.
[0157] The temperature at which the cells are fixed can range from
about 0.degree. to about room temperature. In some embodiments, the
fixation temperature can vary from about 0.degree. C. to about
10.degree. C. In some embodiments, the fixation temperature can
vary from about 0.degree. C. to about 4.degree. C. In one
embodiment, the fixation temperature can be about 4.degree. C.
[0158] The fixation time duration (i.e., time elapsing before the
cell layer (e.g., endothelial cell monolayer) is fixed once a
fixative agent or composition is added) can vary with a number of
factors, including, e.g., but not limited to types, temperature
and/or concentration of the selected fixative agent or composition.
For example, the higher the concentration of a fixative agent or
composition is used, the shorter the fixation time duration can be.
For example, when paraformaldehyde at a concentration of about 4%
is used to fix the cell monolayer (e.g., an endothelial cell
monolayer), the fixation time duration can range from about 15
minutes to about 30 minutes. In one embodiment, the fixation time
duration can be about 20 minutes.
[0159] The inventors have showed that once the cell monolayer
(e.g., endothelial cell monolayer) is fixed, the fixed cell
monolayer (e.g., endothelial cell monolayer) can be stored for a
period of time without significantly reducing its applicability,
e.g., to determine platelet dynamics analysis, as compared to a
freshly fixed cell monolayer or a fixed cell monolayer that has
been stored for a shorter period of time. Accordingly, in some
embodiments, the surface comprising the fixed cell monolayer (e.g.,
fixed endothelial cell monolayer) can have been stored for a period
of time prior use. In some embodiments, the fixed cell monolayer
(e.g., fixed endothelial cell monolayer) can be stored at a
non-freezing temperature. For example, in some embodiments, the
fixed cell monolayer (e.g., fixed endothelial cell monolayer) can
be stored at room temperature. In some embodiments, the fixed cell
monolayer (e.g., fixed endothelial cell monolayer) can be stored at
a temperature of about 4.degree. C. or lower. In some embodiments,
the fixed cell monolayer (e.g., fixed endothelial cell monolayer)
can be stored at a temperature of about 4.degree. C.-10.degree.
C.
[0160] The period of time to store the fixed cell monolayer (e.g.,
fixed endothelial cell monolayer) without significantly reducing
its applicability (i.e., shelf-life of the surface comprising a
fixed cell monolayer (e.g., a fixed endothelial cell monolayer))
can vary with the selected storage temperature. In some
embodiments, the period of time (shelf-life) can be at least about
1 day or longer, including, e.g., at least about 2 days, at least
about 3 days, at least about 4 days, at least about 5 days or
longer. In some embodiments, the period of time (shelf-life) can be
at least about 5 days or longer. In some embodiments, the period of
time (shelf-life) can be at least about 1 week or longer. In some
embodiments, the period of time (shelf-life) can be at least about
2 weeks or longer, including, e.g., at least about 3 weeks, at
least about 4 weeks or longer.
[0161] In some embodiments where the fixed cell monolayer comprises
a fixed endothelial cell monolayer, the fluid sample can comprise a
blood sample, a serum sample, a plasma sample, a lipid solution, a
nutrient medium, or a combination of two or more thereof. In some
embodiments, the fluid sample can comprise at least one type of
blood cells, e.g., red blood cells, white blood cells, and
platelets. In one embodiment, the fluid sample can comprise
platelets, and no red blood cells. In this embodiment, the method
described herein can further comprise removing red blood cells from
the fluid sample (e.g., a blood sample) prior to flowing the blood
sample over the surface.
[0162] Without wishing to be bound by theory, platelet function may
depend upon the presence of Ca.sup.2+ and Mg.sup.2+. Thus, for a
fluid sample comprising a citrated blood sample (where citration of
a blood sample generally quenches the free Ca.sup.2+ and Mg.sup.2+
ions to prevent blood coagulation), addition of Ca.sup.2+ (e.g.,
calcium chloride) and Mg.sup.2+ (magnesium chloride) to the fluid
sample can help restore the native physiological state of the
platelet, e.g., to allow platelet aggregation or coagulation. Thus,
in some embodiments, the citrated blood sample can be added with
Ca.sup.2+ (e.g., calcium chloride) and Mg.sup.2+ (magnesium
chloride) such that the final concentrations reach about 4-12 mM
and 3-10 mM, respectively. However, when a blood sample is
collected in the presence of thrombin blockers to prevent blood
coagulation (e.g., but not limited to heparin, hirudin, EDTA, PPACK
and/or any other anticoagulant), the addition of Ca.sup.2+ (e.g.,
calcium chloride) and Mg.sup.2+ (magnesium chloride) may not be
required.
[0163] In some embodiments, the surface can be a surface of a
fluid-flowing conduit or passageway disposed in a solid substrate.
In some embodiments, the solid substrate can comprise a cell or
tissue culture device, including, e.g., but not limited to a
transwell, a microwell, a microfluidic device, a bioreactor, a
culture plate, or any combinations thereof.
[0164] In some embodiments, the surface can be a solid surface. For
example, in one embodiment, the solid surface can be a wall surface
of a fluid channel, e.g., a microfluidic channel.
[0165] In some embodiments, the surface can be a porous or
gas-permeable surface. For example, in one embodiment, the surface
can be a surface of a gas-permeable membrane. In some embodiments,
the membrane can be configured to separate a first chamber (e.g., a
channel or a compartment) and a second chamber (e.g., a channel or
a compartment) in a cell or tissue culture device.
[0166] In some embodiments, the surface can be disposed in a
microfluidic device. In one embodiment, the microfluidic device can
be an organ-on-a-chip device. Examples of various organ-on-a-chip
devices, e.g., as described in International Patent Application
Nos: WO 2010/009307, WO 2012/118799, WO 2013/086486, WO
2013/086502, and in U.S. Pat. No. 8,647,861, the contents of each
of which are incorporated herein by reference in their entireties,
can be utilized to perform the methods described herein. In one
embodiment, the organ-on-a-chip device can comprise a first channel
and a second channel separated by a membrane. The membrane can be
porous (e.g., permeable or selectively permeable), non-porous
(e.g., non-permeable), rigid, flexible, elastic, or any combination
thereof. In some embodiments, the membrane can be porous, e.g.,
allowing exchange/transport of fluids (e.g., gas and/or liquids),
passage of molecules such as nutrients, cytokines and/or
chemokines, cell transmigration, or any combinations thereof. In
some embodiments, the membrane can be non-porous. In some
embodiments, a first surface of the membrane facing the first
channel comprises a fixed cell monolayer (e.g., a fixed endothelial
cell monolayer) adhered thereon. In some embodiments, a second
surface of the membrane facing the second channel can comprise
tissue-specific cells adhered thereon. In some embodiments, the
membrane can be replaced or embedded with extracellular matrix
proteins (e.g., but not limited to collagen, laminin, etc.). In
some embodiments, the membrane can also comprise smooth muscle
cells and/or fibroblasts.
[0167] By detecting interaction between cells (e.g., blood cells
such as platelets) in the fluid sample and the fixed cell monolayer
(e.g., fixed endothelial cell monolayer), temporal and/or spatial
dynamics of the cells in the fluid sample interacting with each
other and/or to the fixed cell monolayer can be measured. In some
embodiments, the measured temporal and/or spatial dynamics of cell
interaction measured can comprise cell adhesion, cell detachment,
cell translocation, and cell embolization/aggregation. As used
herein, the term "cell adhesion" refers to spatial and/or temporal
adhesion of cells (e.g., platelets) to each other and/or to a fixed
cell monolayer (e.g., fixed endothelial cell monolayer) when the
fluid sample flows over the fixed cell monolayer (e.g., fixed
endothelial cell monolayer). As used herein, the term "cell
detachment" refers to spatial and/or temporal detachment of cells
from cell-cell binding (e.g., between platelets) and/or from the
fixed cell monolayer (e.g., fixed endothelial cell monolayer) when
the fluid sample flows over the fixed cell monolayer (e.g., fixed
endothelial cell monolayer). As used herein, the term "cell
translocation" refers to temporal movement of cells (e.g.,
platelets) from one position to another when the fluid sample flows
over the fixed cell monolayer (e.g., fixed endothelial cell
monolayer). As used herein, the term "cell
embolization/aggregation" refers to spatial and/or temporal binding
of cells (e.g., platelets) to form an aggregate, clump, or embolic
material when the fluid sample flows over the fixed cell monolayer
(e.g., fixed endothelial cell monolayer).
[0168] In some embodiments, the measured temporal and/or spatial
dynamics of cell interaction can comprise binding dynamics of the
cells (e.g., blood cells such as platelets) to the fixed cell
monolayer (e.g., fixed endothelial cell monolayer), binding
dynamics of the cells (e.g., blood cells such as platelets) to each
other, or a combination thereof.
[0169] Depending on cell detection methods, the cells (e.g., blood
cells such as platelets) in the fluid sample can be label-free or
labeled. In some embodiments, the cells (e.g., blood cells such as
platelets) can be label-free. In these embodiments, phase-contrast
or brightfield microscopy can be used to detect the cells when they
are flowing across the surface comprising a fixed cell monolayer.
In some embodiments where the methods described herein are used for
platelet function analysis, the label-free platelets can be
detected by phase-contrast or brightfield microscopy. In some
embodiments where red blood cells may obscure the view, the fluid
sample can be pre-treated to remove red blood cells, or formation
of platelet aggregates can be analyzed by an indirect method, e.g.,
assessment of red blood cell streamlines around a growing platelet
aggregate. Methods for analyzing formation of an aggregate in a
label-free manner are known in the art, including, for example, but
not limited to microscopy and local impedance spectroscopy (a
physical, electrophysiological measurement).
[0170] In some embodiments, the cells (e.g., blood cells such as
platelets) can be labeled. As used herein, the term "labeled"
refers to a cell being manipulated to express or carry a detectable
label, e.g., to facilitate detection of the presence or absence of
the cell. As used herein, the term "detectable label" refers to a
composition capable of producing a detectable signal indicative of
the presence of a target. Detectable labels suitable for the
detection methods that provide spatial and/or temperate information
about cell dynamics (e.g., cell adhesion, cell detachment, cell
translocation, and/or cell embolization/aggregation) described
herein can include any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, magnetic,
optical, chemical means, or a combination of two or more thereof.
In some embodiments, the detectable label can encompass any imaging
agent (e.g., but not limited to, a fluorophore, a nanoparticle,
and/or a quantum dot).
[0171] An exemplary detectable label can comprise a fluorescent
label or fluorophore. A wide variety of fluorescent reporter dyes
are known in the art. Typically, the fluorophore is an aromatic or
heteroaromatic compound and can be a pyrene, anthracene,
naphthalene, acridine, stilbene, indole, benzindole, oxazole,
thiazole, benzothiazole, cyanine, carbocyanine, salicylate,
anthranilate, coumarin, fluorescein, rhodamine or other like
compound.
[0172] In some embodiments, the detectable label can be conjugated
to a cell-targeting moiety. For example, platelets can be labeled
with a detectable label that is conjugated to a platelet-targeting
moiety. Examples of platelet-targeting moieties include, but are
not limited to, platelet endothelial cell adhesion molecule (e.g.,
CD31), antibodies to platelet surface protein (e.g., CD41, CD61,
and/or CD42b).
[0173] Any art-recognized cell detection methods can be used to
detect interaction between cells in the fluid sample and the fixed
cell monolayer. In some embodiments, an imaging-based method can be
used. An exemplary imaging-based method can comprise time-lapse
microscopy, wide-field holography, stereomicroscopes, cameras,
compact mobile devices, or any combinations thereof.
[0174] The fluid sample can be flowed over the surface comprising a
fixed cell monolayer (e.g., fixed endothelial cell monolayer) at a
pre-determined shear rate or flow rate. For example, the fluid
sample can be flowed over the surface at a flow rate that generates
a physiological or pathological wall shear rate. In some
embodiments, the fluid sample can be flowed over the surface at a
flow rate that generates a physiological or pathological arterial
shear rate. For example, the physiological or pathological wall
shear rate can range from about 50 sec.sup.-1 to about 10,000
sec.sup.-1. In some embodiments, the physiological or pathological
shear rate can range from about 100 sec.sup.-1 to about 1,000
sec.sup.-1. In some embodiments, the physiological or pathological
shear rate can range from about 200 sec.sup.-1 to about 900
sec.sup.-1. In one embodiment, the physiological or pathological
shear rate can be about 750 sec.sup.-1. Various methods to flow a
fluid sample over a surface in a chamber are known in the art. For
example, the fluid transport over the surface comprising a fixed
cell monolayer (e.g., fixed endothelial cell monolayer) can be
achieved by syringe pump, capillary driven flow, gravitational flow
and/or pressure-driven flow.
[0175] The fixed cell monolayer (e.g., fixed endothelial cell
monolayer) and the fluid sample can be derived from the same
subject or from different sources.
[0176] As used herein, the term "reactive fluid sample cells"
refers to cells from a fluid sample having a higher frequency
(e.g., by at least about 30% or more) of binding with each other
(e.g., aggregation), and/or with a fixed cell monolayer (e.g.,
adhesion, detachment, and translocation), as compared to control
cells (e.g., healthy cells, or nonstimulated cells).
[0177] As used herein, the terms "treat" or "treatment" or
"treating" refers to both therapeutic treatment and prophylactic or
preventative measures, wherein the object is to prevent or slow the
development of the disease, such as slow down the development of a
blood cell-induced disease or disorder, or reducing at least one
effect or symptom of the blood cell-induced disease or disorder.
Treatment is generally "effective" if one or more symptoms or
clinical markers are reduced as that term is defined herein.
Alternatively, treatment is "effective" if the progression of a
disease is reduced or halted. That is, "treatment" includes not
just the improvement of symptoms or markers, but also a cessation
of at least slowing of progress or worsening of symptoms that would
be expected in absence of treatment. Beneficial or desired clinical
results include, but are not limited to, alleviation of one or more
symptom(s), diminishment of extent of disease, stabilized (i.e.,
not worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment.
[0178] As used herein, the term "platelet dysfunction" refers to
abnormal platelet behavior, as compared to healthy platelets. In
one embodiment, platelet dysfunction can be caused by increased
adhesion to an endothelium (e.g., by at least about 30% or more),
as compared to healthy platelets. In one embodiment, platelet
dysfunction can be caused by abnormal detachment from other
platelets and/or from an endothelium (e.g., by at least about 30%
or more), as compared to healthy platelets. In one embodiment,
platelet dysfunction can be caused by abnormal translocation (e.g.,
by at least about 30% or more), as compared to healthy platelets.
As used herein, the term "abnormal translocation" refers to a
platelet that gets activated in one location and deposits at
another location to form a clot or cause inflammation response. For
example, thromboembolism can be considered as abnormal
translocation. In one embodiment, platelet dysfunction can be
caused by increased aggregation between platelets (e.g., by at
least about 30% or more), as compared to healthy platelets.
[0179] Generally, in reference to one aspect, a simple microfluidic
device is lined by a chemically preserved human endothelium that
retains its ability to support thrombus formation and platelet
adhesion as blood flows through its channels at an arterial shear
rate. This biomimetic device demonstrates the potential practical
value for laboratory and point-of-care settings by showing that it
can be more rapid and reliable than standard aggregometry or
similar collagen-coated microfluidic devices.
[0180] Generally, in reference to another aspect, a lung-on-a-chip
microfluidic device reconstitutes critical functional aspects of
intravascular pulmonary thrombus formation and platelet-endothelial
dynamics in the vicinity of an endotoxin or cytokine stimulated
primary human alveolar epithelium. The lung-on-a-chip microfluidic
device is perfused with human whole blood in a bioengineered
vascular lumen. Additionally, based on inherent dynamic complexity
and spatiotemporal heterogeneity in the way platelets interact with
the vessel wall and initiate thrombus formation, a method is
directed to a large-scale real-time fluorescence image acquisition
and a statistical algorithm for quantifying platelet-endothelial
dynamics during endothelial dysfunction.
[0181] Combining the supporting quantitative analysis and the in
vitro organ-level functional microfluidic device, the pulmonary
thrombosis-on-chip model has been extended to evaluate the
cytoprotective affect of a potential protease activator receptor
("PAR-1") inhibitor, termed parmodulin 2 ("PM2"). Thus, the results
lead to the proposition that this inhibitor is a potential
anti-thrombotic and anti-inflammatory therapeutic drug for human
patients to treat disorders that lead to acute lung thrombosis.
I. Fixed Cells--Materials and Methods
[0182] A. Microfluidic Device Design and Treatment
[0183] By way of example, a microfluidic device is fabricated using
photolithography followed by soft lithography with
polydimethysiloxane ("PDMS"). According to this example, six
microfluidic devices are placed on a single PDMS mold of the size
of a standard glass slide, e.g., 75.times.25 millimeters ("mm").
The microfluidic devices are first exposed to oxygen plasma for 30
seconds using, for example, a PE-100 benchtop plasma system from
Plasma Etch. Then, the microfluidic devices are treated with 1%
(3-aminopropyl)-trimethoxysilane ("APTMS"), from Sigma, in 100%
anhydrous ethanol for ten minutes. After subsequent rinsing with
70% and 100% ethanol, the microfluidic devices are dried and type I
collagen from rat tail, e.g., 100 micrograms/milliliter (".mu.g/ml)
from Corning, is introduced in microchannels of the microfluidic
devices.
[0184] The microfluidic devices are left overnight at 37.degree. C.
in a 5% carbon dioxide ("CO.sub.2") incubator, after which they are
rinsed with Endothelial Growth Medium-2 ("EGM-2") from Lonza. Human
Umbilical Vein Endothelial Cells ("HUVEC") from a mixed donor, by
Lonza, are kept in culture and suspended at 12.5 million
cells/milliliter ("mL") in EGM-2, after confluence. The suspension
is introduced into the collagen-coated microchannels, after which
the microfluidic devices are incubated upside down for 20 minutes.
A fresh HUVEC suspension is then introduced in the microchannels,
after which the microfluidic devices are incubated for eight hours
to promote cell attachment across the channel. The microchannels
are, then, rinsed with EGM-2, sometimes containing a freshly
prepared solution of tumor necrosis factor ("TNF-.alpha."), which
is a recombinant from E. coli from Sigma. Antibodies against
intercellular adhesion molecule-1 ("ICAM-1") and vascular adhesion
molecule-1 ("VCAM-1"), tissue factor (from Santa Cruz), VWF (from
Abcam) and VE-Cadherin (from Santa Cruz) were perfused into the
microfluidic devices after fixing the endothelium with 2%
paraformaldehyde for ten minutes, incubating the endothelium for
three hours, and counterstaining with a secondary fluorescent IgG
antibody for three hours.
[0185] B. Blood Samples and Human Subjects
[0186] Human blood (e.g., received from Research Blood Components,
Cambridge, Mass.) is acquired in 3.2% sodium citrate tubes and is
used within 5 hours of blood draw, to prevent pre-analytical
effects on platelet function. Institutional review board ("IRB")
approval is obtained for use of discarded blood samples. Subjects
are selected from among patients who re taking antiplatelet
medication. A total of 11 samples are used for analysis, from which
8 patients re on aspirin alone and 3 re on aspirin and clopidogrel
(Plavix).
[0187] C. Blood Perfusion
[0188] 500 microliters (".mu.L") of whole blood are pipetted into a
fluid reservoir fitted to one end of a microchannel. Platelets are
labeled with human CD41-PE antibody (e.g., 10 .mu.L/mL from
Invitrogen) that is directly added to the blood samples and
incubated at room temperature for 10 minutes. Fluorescently labeled
fibrinogen (e.g., 10 .mu.g/mL from Life Sciences) is added, if
required. Blood is pulled through the device (e.g., 30 .mu.L/min)
via tubing at an outlet using a syringe pump (e.g., PHD Ultra.TM.
CP, Harvard Apparatus), resulting in an arterial shear rate of 750
s.sup.-1. After two minutes, blood is supplemented with 100
millimolars ("mM") calcium ("CaCl.sub.2") and 75 mM magnesium
("MgCl.sub.2") to support coagulation-activated blood clotting
(e.g., 100 .mu.L/mL).
[0189] D. Image Acquisition and Analysis
[0190] Platelets are visualized using time-lapse fluorescence
imaging (e.g., 20.times., NA 0.4). A time series of a 10-frame
panorama (e.g., 6 mm long.times.0.665 mm wide region of a
microchannel), at a lapse of every 30 seconds is recorded. The
resulting image stack is maximum intensity projected along time,
thresholded, segmented, and cropped to a central 200 microns
(".mu.m") of the channel width for analysis. Finally, platelet
coverage is computed from the binary image as the ratio of bright
pixels to the total number of pixels in the image.
[0191] E. Light Transmission Aggregometry ("LTA")
[0192] The LTA is performed in accordance with manufacturer
instructions. For example, the LTA uses adenosine diphosphate
("ADP") 10 .mu.M and collagen 2 .mu.g/mL from Chrono-Log.
[0193] F. Statistical Analysis
[0194] All data is presented as mean.+-.standard error (SEM).
Two-tailed P values are obtained from a statistical t-test or one
way ANOVA using GraphPad Prism V6.
II. Fixed Cells--Results and Discussion
[0195] A. Formation and Evaluation of a Chemically Preserved
Endothelium
[0196] In reference to FIG. 1 and the above example, a microfluidic
device 100 is engineered that contains a rectangular microchannel
102. By way of example, the microchannel 102 is 400 .mu.m wide, 100
.mu.m high, and 2 centimeters ("cm") long. The microfluidic device
100 has a blood inlet reservoir 104, followed by the straight
microchannel 102 that ends at an outlet 106. A syringe pump is
attached to the outlet 106 to pull the blood from the microchannel
102.
[0197] In reference to FIG. 2, a microfluidic device 200 is made of
PDMS and has six similar and independent microchannels 202 that are
provided on a single chip. The microchannels 202 are similar to or
identical with the microchannel 102 illustrated in FIG. 1.
According to one example, the chip 200 is 15 cm long and is
fabricated on a glass slide.
[0198] In reference to FIGS. 3 and 4A-4C, the inner surface of the
rectangular microchannel 102 is coated with collagen and, then, is
cultured with HUVECs to create a tube lined by a continuous,
confluent endothelial cell monolayer. Specifically, FIG. 3
illustrates a fluorescence micrograph that shows the entire
microchannel covered with HUVECs stained with VE-cadherin junction
marker (scale bar--1 mm). FIGS. 4A-4C illustrate confocal
immunofluorescence microscopic images showing a section of the
microchannel 102 with HUVECs when viewed from above in the xy plane
(FIG. 4A), and reconstruction of cross-sectional views from the
front yz plane (FIG. 4B) and the side xz plane (FIG. 4C). This
demonstrates full coverage of all microfluidic microchannel walls.
In this example, the microfluidic device 100 includes VE-Cadherin,
nuclear DAPI, with a scale bar of 200 .mu.m.
[0199] Multiple endothelial adhesion molecules are involved in the
recruitment of blood cells and platelets in thrombosis in vivo. In
previous studies, treatment of a living endothelium cultured in
microfluidic channels with TNF-.alpha. results in an increase in
expression of surface adhesion molecules, such as ICAM-1 and VCAM-1
within four hours after addition. To explore whether a fixed
endothelium retains expression of these and other surface molecules
that could potentially exacerbate thrombosis, the endothelium is
first activated in the device by adding increasing doses of
TNF-.alpha., e.g., 0, 5, and 100 nanograms/millliter ("ng/ml"), for
approximately 18 hours. The endothelium cells are fixed with 4%
paraformaldehyde in phosphate-buffered saline ("PBS") for 15
minutes at room temperature, are rinsed three times with PBS, and,
then, stored in PBS at 4.degree. C. in a humid environment.
[0200] In reference to FIGS. 5-8, graphs show fluorescence
(normalized by the untreated endothelium) measured after
immunostaining the fixed endothelium, which continues to exhibit a
dose-dependent increase in ICAM-1 (FIG. 5), VCAM-1 (FIG. 6), VWF
(see. 7), or tissue factor (FIG. 8). In the graphs, *P<0.05
versus untreated and n=3. These results indicate that the fixed
endothelium retains expression of multiple molecules that mediate
adhesion of blood cells and platelets after activation with
TNF-.alpha., and induce clotting.
[0201] B. Fibrin and Platelet Function Analysis Using a Fixed
Endothelium
[0202] To explore whether the fixed endothelium in the microfluidic
devices retains the ability to promote hemostasis, recalcified
citrated whole blood (coagulation activated) is immediately
perfused through a microfluidic channel lined by the fixed
endothelium and preserved for 24-36 hours. Platelet adhesion is
analyzed for 15 minutes of flow.
[0203] Referring generally to FIGS. 9-11B, platelet coverage and
fibrin formation is illustrated on a fixed endothelium in the
microfluidic device 100. When blood from a healthy donor is flowed
over an endothelium that is fixed without prior treatment with
TNF-.alpha., there is virtually no platelet adhesion on the
surface, as would be expected for a healthy endothelium. In
contrast, when microfluidic devices are used with endothelium that
is treated with increasing doses of TNF-.alpha. prior to fixation,
a dose-dependent increase in platelet surface adhesion to the
endothelial layer is observed.
[0204] Referring more specifically to FIG. 9, representative
maximum intensity projection micrographs show fluorescently labeled
platelets adhering to a chemopreserved endothelium in a TNF-.alpha.
does-dependent manner (scale bar--100 .mu.m). Referring to FIG. 10,
a graph shows platelet coverage when blood is perfused inside the
microchannel 102 that is lined with a living or fixed endothelium,
which has been stimulated by TNF-.alpha. before fixation. Of note,
no significant difference is observed in platelet adhesion when
comparing living versus chemopreserved endothelium, with or without
treatment with TNF-.alpha.. For example, P>0.05 at each
TNF-.alpha. concentration (n=4, *P<0.05). Referring to FIGS. 11A
and 11B, fluorescent micrographs show fibrin that is formed along
with platelet aggregates on a fixed endothelium, which has been
pretreated with TNF-.alpha. (5 ng/ml) and perfused with recalcified
citrated whole blood (FIG. 11A--scale bar--200 .mu.m; FIG.
11B--scale bar--20 .mu.m).
[0205] These results confirm that the fixed surface of the
endothelium retains its pro-thrombotic function after fixation.
Furthermore, when perfusing whole blood containing fluorescently
labeled fibrinogen, the thrombi also contains a significant amount
of fibrin if the endothelium was pre-treated with TNF-.alpha.
before fixation. This further confirms that the preserved
endothelial surface also retains its ability to activate the
coagulation cascade. The morphology of these thrombi also appear
similar to that of thrombi formed on living endothelium in vivo,
and significantly different from bare collagen-coated flow.
Together, these results demonstrate that the fixed endothelium is
capable of reproducing physiologically-relevant thrombus formation
in our microfluidic device.
[0206] C. Potential Clinical Utility of the Device Lined with Fixed
Endothelium
[0207] In accordance with another exemplary embodiment, a
microfluidic device containing a fixed endothelium is used to
detect antiplatelet drug effects in healthy donors and patients
taking antiplatelet medication. For example, the cultured
endothelium is pretreated with a physiologically relevant dose of
TNF-.alpha. (e.g., 5 ng/mL). The microfluidic device contains the
fixed activated endothelium with whole blood from a healthy donor
containing 0 to 100 .mu.g/mL (e.g., a clinical range .about.1-10
.mu.g/mL) of the antiplatelet GP IIb/IIIa antagonist, abciximab
(ReoPro.RTM.).
[0208] Referring to FIG. 12, a graph illustrates platelet coverage
on a fixed endothelium that is pretreated with TNF-.alpha. when
blood samples, which contain different doses of the drug abciximab,
are perfused through a microfluidic device (such as the
microfluidic device 100 illustrated in FIG. 1). Thus, when the
microfluidic device is perfused, a dose-dependent inhibition of
platelet adhesion is observed, with optimal effects observed at 10
.mu.g/mL or higher. This is consistent with previous studies using
flow cytometric analysis.
[0209] Referring to FIG. 13, a graph illustrates light transmission
aggregometry of blood samples containing different doses of
abciximab using either ADP or collagen as an agonist (n=4). Thus,
in contrast to the results shown in FIG. 12, all concentrations of
the drug abciximab produce virtually complete inhibition of
platelet aggregation (e.g., no dose dependence) as detected using
LTA, regardless of whether ADP or collagen was used as an
agonist.
[0210] Referring to FIG. 14, a graph illustrates platelet coverage
when blood samples containing different doses of the drug abciximab
are perfused through collage-coated microfluidic devices (n=4).
While there appears to be a small suppressive effect on platelet
adhesion when the same blood samples are flowed through an
acellular collagen-coated flow chamber, the sensitivity is
extremely low and the differences between abciximab doses are not
statistically significant.
[0211] Thus, the microfluidic device containing the fixed
endothelium provides an optimally sensitive measure of platelet
function, with a higher dynamic response across a range of
abciximab concentrations than existing platelet function assays.
These results suggest that a fixed endothelialized microfluidic
device is likely useful in monitoring antiplatelet regimens in
patients and has functional advantages over acellular conventional
assays. These results also indicate that the fixed surface of the
endothelium retains its ability to modulate platelet interactions
via a GPIIb/IIIa pathway, the target of abciximab, which is
involved in multiple thrombotic and vascular processes.
[0212] Referring to FIG. 15, a graph illustrates platelet coverage
on a fixed endothelium that has been pretreated with TNF-.alpha.
when blood samples from healthy donors, versus subjects treated
with antiplatelet drugs, are perfused through microfluidic devices
(n=11). Thus, whole blood is perfused from subjects who are regular
users of antiplatelet drugs, with the subjects showing a
significant reduction in platelet aggregation when tested using the
microfluidic devices, in comparison to healthy donors.
[0213] Referring to FIG. 16, a graph illustrates light transmission
aggregometry of healthy versus antiplatelet treated blood samples
using ADP or collagen as an agonist (n=11). Thus, while similar
results are obtained using conventional LTA, a microfluidic assay
in accordance with the present disclosure requires a significantly
reduced time period to complete (e.g., only 15 minutes) relative to
the much longer time period for an aggregometry test (which must
account for sample preparation time).
[0214] Referring to FIG. 17, a graph illustrates platelet coverage
when healthy versus subject blood samples are perfused through
collagen-coated microfluidic devices (n=11; *P<0.05). Thus,
platelet inhibition in these subjects is not reliably detected on a
collagen-coated flow chamber as there is no significant difference
in platelet coverage between normal controls and subjects.
Accordingly, the embodiments described above demonstrate that a
microfluidic device containing fixed endothelium is potentially
applicable in point-of-care settings.
[0215] One benefit of a microfluidic device, which contains human
endothelial cells that are chemically preserved by fixation, is
that it can be stored, shipped, and used when required, either in a
laboratory setting or in point-of-care settings. Another benefit of
such a microfluidic device is that it is a functional assay used to
evaluate platelet aggregation and inhibition with drugs in blood
samples of patients. Yet another benefit of such a microfluidic
device is that it provides an assay with increased sensitivity than
existing assays, and is helpful in rapid analysis of platelet
function and hemostasis while incorporating contributions from the
endothelium and dynamic blood flow.
[0216] Although the fixed endothelium may lose some of its live in
vivo functions (e.g., release of bioactive messengers like nitric
oxide), and the exact mechanism by which the surface promotes
platelet aggregation and thrombosis is most likely multi-factorial,
the above-discussed results suggest that for a period of 15 minutes
of blood flow, the fixed endothelium retains its ability to prevent
blood clotting under unstimulated conditions. The results further
suggest that the fixed endothelium promotes platelet aggregation
and thrombosis when pretreated with TNF-.alpha. prior to fixation.
Notably, the qualitative and quantitative pro-thrombotic and
pro-coagulant responses of the fixed endothelium closely mimic
those of the living endothelium, suggesting that the fixed
endothelium also permits the study of thrombus formation on a
surface that mimics an inflamed endothelium, such as might be found
in an atherosclerotic plaque.
III. Algorithm--Results
[0217] A. Morphological and Quantitative Analysis of Thrombus
Formation
[0218] Referring to FIG. 18, a microfluidic device 300 in the form
of a bioengineered microfluidic blood vessel contains cultured
endothelial cells 302 on all walls of a microchannel 304 (also
referred to as a vascular chamber). According to one embodiment,
the microfluidic device 300 is similar to or identical with any of
the microfluidic devices 100, 200 described above. The vessel 300
and endothelial cells 302 are intended to mimic morphology of a
blood clot seen in vivo. Thus, whole blood (containing
fluorescently labelled platelets) is perfused through the
microfluidic device 300.
[0219] The morphology of thrombus and platelet-endothelial
dynamics, which occurs in the microfluidic device 300, is
characterized via an imaging and quantitative analysis technique.
Specifically, the technique characterizes the morphology as it may
occur in vivo and as a result of endothelial cells activation,
blood cells, and shear stress. For example, imaging of recalcified
citrated whole blood, which contains labelled platelets perfused
inside a small section of a vascular chamber 304 (no epithelial
cell culture), platelet adhesion on a bare collagen surface occurs
rapidly, firmly, and increasing steadily over a timespan in the
range of about 10 minutes (e.g., 2.5-12.5 minutes).
[0220] Referring to FIG. 19, fluorescence micrographs depict a
section of the imaged microchannel 304 showing platelet
accumulation (left to right) on collagen, a healthy blood vessel,
and a TNF-.alpha. stimulated vessel. This imaging is consistent
with past observations and is reminiscent of formation of a
hemostatic plug under vascular injury. In contrast, when the lumen
chamber 304 is covered with a continuous living endothelial
monolayer over collagen, very little platelet interactions and
aggregate formation occur over the course of an experiment, much as
what is observed in blood flowing in a healthy human blood vessel.
However, when endothelial cells are stimulated with an inflammatory
cytokine tumor necrosis factor (e.g., TNF-.alpha.; 100 ng
ml.sup.-1) prior to blood flow, platelet adhesion again occurs.
Nevertheless, the morphology of the thrombus is clearly distinct
from aggregates formed on the collagen surface.
[0221] Referring to FIG. 20, fluorescence micrographs depict a
section of the imaged microchannel 304 showing platelet
accumulation after 4 minutes of laser-induced injury on a mouse
cremaster arteriole (scale bar--.mu.m 25). The typical size of
aggregates on activated endothelium is visibly larger and,
interestingly, the size, shape, and organization of the thrombi
formed on the activated endothelium in this in vitro model
correlates well with what is observed in a mouse model of
laser-induced thrombosis in vivo. This confirms the patency of the
in vitro setup in reproducing physiologically-relevant thrombus
formation, which is missing in simple collagen-coated devices.
[0222] Referring to FIG. 21, fluorescent micrographs of a large
section of the vascular chamber 304 shows intravascular thrombus
formation in collagen (top image), and TNF-.alpha. stimulated
endothelium in a dose dependent manner (bottom three images). The
scale bar is 100 .mu.m. The micrographs are helpful in identifying
quantitative parameters for a comparative and cumulative analysis
of a large number of platelet-endothelial interactions that occur
over a long region in the microfluidic device 300.
[0223] Specifically, the analysis includes a method to quantitate
platelet function in flow chambers and microfluidic devices that
has been primarily limited to analyzing platelet adhesion on bare
collagen surfaces. To analyze platelet-endothelial dynamics that
will act as a robust readout of physiologically-relevant clotting,
an automated imaging program creates an image time-series, K(x, y,
t), containing a 10-frame panorama in space. Images are acquired at
a frame rate of 2 panoramic images per minute, for a total time of
15 minutes. A non-dimensional stochastic index is derived to
quantitate platelet endothelial dynamics ("P-E"), which is the
interpercentile range of a coefficient of variance of the image
time-series:
P-E=range(CV(K(x,y,t) (1)
A feature of this analytical readout is that instead of an
"ensemble averaging," the method incorporates the cell-surface
interactions at the pixel level and quantitates statistical
"dispersion" of interactions.
[0224] Referring to FIG. 22, a graph illustrates ICAM-1 expression
on the endothelial cells after stimulation with TNF-.alpha..
Specifically, the graph illustrates testing of the sensitivity of
parameter (P-E) in response to changes in endothelial activation,
and the stimulation of the endothelial vessel with various doses of
TNF-.alpha., which result in a dose-dependent surface expression of
adhesion molecule ICAM-1 (n=3).
[0225] Referring to FIG. 23, a graph illustrates a sensitivity
analysis of the platelet endothelial dynamics algorithm, showing
that in conditions of hemostasis (e.g., vascular injury/collagen or
healthy endothelium), the dynamics are near absent. However, the
dynamics increase in a TNF-.alpha. dose dependent manner. The
platelet endothelial dynamics are also sensitive to applied shear
rate (e.g., n=3, *P<0.05). Thus, a dose-dependent effect in P-E,
shows that the method is sensitive to vasculopathy induced
thrombosis (e.g., n=4).
[0226] In fact, unlike platelet adhesion (such as illustrated in
FIG. 40), the P-E on a bare collagen surface is extremely low. This
shows that the present method distinguishes between the conditions
of hemostasis (e.g., in a healthy vessel or vascular injury) and
thrombosis due to inflammation. Furthermore, shear stress is a
major determinant, for example, of endothelial function, blood
rheology, platelet activation, or immune function, which, together,
can alter thrombosis.
[0227] The P-E parameter is also sensitive to applied shear stress,
and is indicative of a higher tendency to form platelet-rich
thrombi and platelet-endothelial interactions, as shear is
increased (see FIG. 22). In contrast, area-averaged platelet
adhesion is not sensitive to shear (see FIG. 40). Notably, a CV
colormap of a single thrombus formed in a laser injured mouse in
vivo, shows high reactivity at the boundary compared to the central
core, which is also observed in individual thrombi that are formed
in a microfluidic device, in vitro (see FIG. 41).
[0228] Interestingly, these regional heterogeneities in the
thrombus that are revealed in accordance with the method described
above, have also been confirmed by computational studies and also
in vivo, showing that a thrombus consists of a stable core region
surrounded by reactive unstable shell. Furthermore, the method
described above shows that the platelet-endothelial dynamics (P-E)
parameter is a robust parameter that is applicable to quantitate
platelet function and thrombosis, both in vitro and in vivo, where
the endothelial function is also included.
[0229] B. Engineering of the Pulmonary Thrombosis-On-Chip
[0230] Referring generally to FIGS. 24-26, to model
physiologically-relevant pulmonary hemostasis and thrombosis, a
lung-on-a-chip device not only allows co-culture of lung epithelial
and endothelial cells in the presence of physiological relevant
shear, but also includes primary human cells and a more functional
arterial blood vascular lumen in which human whole blood can be
perfused. Referring specifically to FIG. 24, a conceptual schematic
of a human lung 400 shows that the alveoli 402 interacts with
neighboring blood vessels 404 during hemostasis or pulmonary
dysfunction. Referring specifically to FIGS. 25 and 26, a
microfluidic device 500 contains two PDMS compartments 502 (which
include a top compartment 502A and a bottom compartment 502B)
separated by a thin porous membrane 504 that reproduces the
microarchitecture of the alveolar-capillary interface. The
microfluidic device 500, according to this example, is in the form
of a lung-on-a-chip device. The top compartment 502A is cultured
with human primary alveolar epithelial cells 506 and the entire
bottom chamber 502B is lined with human endothelial cells 508
forming a lumen. Whole blood is perfused through the bottom chamber
502B and thrombus formation is visualized using fluorescence
microscopy from the bottom. Optionally, or alternatively, the
compartments 502A, 502B are in the form of channels.
[0231] Referring to FIG. 27, visual stacks of confocal micrographs
show junctional structures, after twelve days of co-culture, of a
single layer of the primary alveolar epithelium 506 at the top
chamber 502A (stained with e-cadherin) and endothelial monolayers
508 covering either side of the lower chamber 502B (stained with
ve-cadherin), through which blood perfusion takes place. The scale
bar for the top and bottom images of FIG. 27 is 50 .mu.m, and the
scale bar for the middle image is 250 .mu.m.
[0232] Thus, in the alveolar chamber 502A, human primary alveolar
epithelial cells ("AE") are cultured and in the blood vessel
chamber 502B, the entire chamber 502B is cultured with human
endothelial cells (.mu.BV) for a total of 12 days, thus creating an
organ-level functional device 500 where the epithelial,
endothelial, and blood cell interactions are visualized and
analyzed in real-time, in the presence of whole blood flow and
conditions that mimic thrombus formation in vivo. Notably, this
lung-on-a-chip device 500 contains co-culture of healthy human
primary alveolar cells and healthy endothelial cells that show
barrier integrity and intact junctions even after 12 days of living
culture, across the entire length and breadth of the device 500.
Accordingly, in the state of hemostasis (e.g., healthy cell culture
and perfusion of healthy blood), perfuse recalcified citrated whole
blood (coagulation activated) is perfused for up to 20 minutes
without any observed platelet adhesion or clotting inside the lumen
of the lung-on-chip device 500. This confirms the formation of a
healthy organ-level functional microfluidic device 500 that is
capable of resembling the state of hemostasis.
[0233] Referring to FIG. 28, a chart shows vascular ICAM-1 measured
after TNF-.alpha. stimulation relative to untreated cells in the
presence of AE (e.g., n=3). Specifically, upon stimulation of the
lung-on-chip microfluidic device 500 with TNF-.alpha. on the
alveolar compartment 502A containing AE, the vascular ICAM-1
expression increases in a dose-dependent manner, reproducing
endothelial stimulation as has been previously observed.
[0234] Referring to FIG. 29, and correspondingly with respect to
the chart of FIG. 28, the platelet-endothelial dynamics in the
microfluidic device 500 follows a similar trend as ICAM-1.
Specifically, a chart shows platelet-endothelial dynamics in an
untreated vs TNF-.alpha. stimulated lung-on-a-chip device 500 (n=3.
*P<0.05).
[0235] Referring to FIG. 30, at the end of the assay, due to
vascular activation, significant amount of clots are observed. The
clots are constituted by platelets and fibrin localized within the
vascular compartment 502B of the lung-on-a-chip device 500.
Specifically, fluorescent micrographs showing platelets (left),
fibrin (middle), and merged (right) on an endothelial surface, when
stimulated by TNF-.alpha. (scale bar--100 .mu.m). This confirms
that the lung-on-a-chip device 500 is sensitive to the
pro-inflammatory effect of the TNF.alpha.. The epithelial
stimulation, which can result in signalling from the epithelial
side to the endothelium, promotes activation of the endothelial
cells and blood cells, such as, platelets, finally causing
intravascular thrombus formation.
[0236] C. Lipopolysaccharide ("LPS") Induced Inflammation and
Thrombosis
[0237] Referring generally to FIGS. 31-35, an evaluation is
directed to more complex epithelial-endothelial-blood cell
interactions and to better define the link between local
inflammation and thrombosis. The evaluation includes stimulating a
microfluidic device on the alveolar epithelial cells (AE) side with
a lipopolysaccharide (LPS) endotoxin, and comparing P-E when the
stimulation occurs in the presence or absence of the alveolar
epithelial cells (AE). The microfluidic device is any of the
microfluidic devices described above (e.g., microfluidic device
300, lung-on-a-chip device 500, etc.).
[0238] Referring specifically to FIG. 31, a chart shows vascular
ICAM-1 that is measured after LPS stimulation relative to untreated
cells in the presence or absence of the alveolar epithelial cells
(AE) (n=3). When the blood vessel alone is stimulated with LPS for
2 hours, it results in no significant increase in ICAM-1, and the
endothelium is inflamed only when the LPS stimulation occurs over
the AE.
[0239] Referring specifically to FIG. 32, platelet-endothelial
dynamics measured in the microfluidic device, in the presence or
absence of the alveolar epithelial cells (AE), are either left
untreated or are stimulated with various doses of LPS (n=3,
*P<0.001). Thus, the P-E is near absent when the lung-on-a-chip
device is left untreated or is treated with LPS in the absence of
AE. Furthermore, the P-E significantly increases when the
stimulation occurs over the AE.
[0240] Referring specifically to FIG. 33, representative
fluorescence micrographs show platelet aggregates and fibrin at the
end of blood perfusion through the microfluidic device. The
microfluidic device contains and compares untreated and LPS
stimulation, in the presence or absence of AE (scale bar--100
.mu.m). Thus, at the end of blood perfusion in the LPS-stimulated
microfluidic device containing AE, large platelet aggregates are
formed along with significant fibrin in the lumen. This
demonstrates in vitro that in situ thrombosis in the vascular lumen
is caused by an LPS-directed epithelial injury.
[0241] Referring specifically to FIG. 34, a chart shows barrier
permeability measured after LPS stimulation, relative to untreated
cells in the presence or absence of the alveolar epithelial cells
(AE) (n=1 or 2). Thus, the trend that occurs due to the epithelium
is further confirmed when the barrier permeability is measured.
[0242] Referring specifically to FIG. 35, representative confocal
micrographs show gap junctions under no treatment or LPS treatment,
in the presence of a blood vessel alone or with epithelium (AE).
The visualized gap junctions are not affected when the blood vessel
is stimulated with LPS, but significantly increase in the presence
of AE.
[0243] Referring generally to FIGS. 36 and 37, the technique
described above in reference to FIGS. 31-35 has been further
applied to a laser injury induced thrombus formation in vivo in a
cremaster arteriole and a cremaster vein of a mouse, along with a
systemic delivery of LPS. When a mouse is injured with laser,
significant clotting is observed in both the artery and vein, along
with increase in P-E. The increase in P-E is further exacerbated
when LPS is administered additionally. However, administration of
LPS alone does not induce any platelet-endothelial
interactions.
[0244] Referring specifically to FIG. 36, fluorescent micrographs
show evolution of blood clots (left to right) in a cremaster artery
of the mouse. The cremaster artery is left untreated, laser injured
or after systemic injection of LPS (scale bar: 25 .mu.m).
[0245] Referring specifically to FIG. 37, a chart shows
platelet-endothelial dynamics computed on fluorescent time-series
of platelets. The platelets adhere to a cremaster artery or a vein
of a mouse (n=3, *P<0.05).
[0246] Referring to FIG. 42, histological sections show sections of
a mouse lung with clots. The sections of the mouse lung are left
untreated or treated with LPS. Thus, in contrast to the results
described in reference to FIGS. 36 and 27, the histological
analysis of the mouse lung shows that the LPS injection results in
lung injury and pulmonary thrombosis. This shows that either an
exogenous stimulation (e.g., a laser injury) or possibly an
epithelium (e.g., in a lung), are likely essential to platelet
adhesion and thrombosis (as also recreated in vitro) in the
microfluidic device. Therefore, an LPS-stimulated lung-on-a-chip
device is potentially a robust model for testing potential
antithrombotic and anti-inflammatory drug candidates, in an
organ-level functional environment of pulmonary
epithelial-endothelial-blood cell signalling.
[0247] D. Analysis of Cytoprotective Effect of PAR-1 Inhibitor
[0248] Referring to FIG. 38, an illustration shows a microfluidic
device 600 that contains alveolar epithelial cells (AE) 602 treated
with LPS 604 and a vessel 606 treated with parmodulin (PM2) 608 to
inhibit thrombosis due to lung injury. Thus, the microfluidic
device 600 is stimulated on the AE side 602 with LPS and is
perfused with blood 610 in the vessel 606. The microfluidic device
is similar to or identical with one or more of the microfluidic
devices described above. In response to the stimulation, no
significant clotting occurs and P-E occurs PM2 is added to the
endothelial cell culture medium.
[0249] Referring to FIG. 39, a chart shows platelet-endothelial
dynamics that are measured in the microfluidic device 600
containing AE cells (n=3. *P<0.05,**P<0.01). Additionally,
adding PM2 to blood 610 alone also results in the reduction of P-E
as it prevents PAR-1 activation of platelets. Furthermore, adding
PM2 to both endothelium and platelets completely inhibits the P-E
and thrombotic activity in this LPS-stimulated alveolar
microfluidic device 600.
[0250] As such, the approach described in reference to FIGS. 38 and
39 show a cytoprotective and anti-thrombotic effect of PM2 in a
physiologically-relevant model of acute lung dysfunction. Thus, the
approach provides a beneficial drug candidate for intervention in
diseases that cause pulmonary thrombosis. Notably, the approach
demonstrates the strength of the in vitro microfluidic device 600,
allowing visualization and quantitative analysis of organ-level
interactions in real-time, including the epithelium (e.g.,
stimulated with LPS), endothelium (e.g., protected by PM2), and
whole blood cells (e.g., coagulation and platelet function). A
similar experiment in vivo is extremely difficult to perform, if
not impossible.
IV. Algorithm--Discussion
[0251] Generally, a salient feature of pulmonary organ-on-chip
microfluidic technology is that it permits perfusion of human whole
blood in its native state (e.g., recalcified after anticoagulation
in sodium citrate) at any desirable shear rate. Thus, the
microfluidic technology provides a significantly more
physiologically-relevant in vitro platform to study and analyze
intravascular clotting of a lung organ.
[0252] Additionally, by harnessing the full potential of modern
automated fluorescence microscopy and mathematical algorithms that
are designed to quantitate thrombus formation occurring in
real-time inside a microfluidic device, platelet-surface
interactions are assessed over large spatiotemporal scales. The
approaches described above and below show that the integrated
interplay between platelets, thrombi, a vessel wall, blood-borne
factors, and flow dynamics are analyzed in the integrated
system-level assay. Significantly, pulmonary clot formation is
caused by endothelial activation that occurs in a microfluidic
device in vitro and correlates to a mouse laser injury model in
vivo, both in terms of morphology and regional heterogeneity. Thus,
the described microfluidic device and analytical methods
potentially act as a valuable tool for analyzing organ-level
tissue-tissue interactions under pathophysiological conditions that
are relevant for thrombosis and platelet research.
[0253] Furthermore, by incorporating primary alveolar epithelial
cells that are co-cultured along with endothelial cells for up to 2
weeks, and maintaining physiological junction integrity, a
limitation of previous lung-on-a-chip devices (which contain tumor
derived cell lines) has been overcome. The present
physiologically-relevant microfluidic device allows the finding
that the epithelial cells can make a direct contribution to
thrombosis, when stimulated by LPS. This finding is very difficult
to show in vivo in real-time, as individual tissue and cellular
compartments inside an organ cannot be individually regulated, and
blood flow inside lung vessels cannot be observed over a large
section.
[0254] Nevertheless, using the laser injury mouse model, it is
demonstrated that LPS does not cause thrombosis in a cremaster
artery or vein, but causes clotting in the lung. This is somewhat
similar to in vitro observations, where LPS stimulation of
endothelium alone does not cause thrombus formation, but, instead,
LPS stimulation of epithelium results in vascular dysfunction and
rapid clotting.
[0255] The described microfluidic devices and methods help unravel
the cytoprotective and anti-thrombotic effect of a novel PAR-1
antagonist (PM2) in the setting of an acute lung injury and whole
blood perfusion. The findings encourage the pharmaceutical industry
to further test antithrombotic drugs using the described humanized
platform, having the potential to cause vasculopathy and bleeding
as a major toxicity (which is also very difficult to study in
vivo). Overall, the described pulmonary thrombosis-on-a-chip
microfluidic device may be further beneficial in finding potential
applications in a variety of settings relevant for thrombosis
research, e.g., toxicology, drug screening, and diagnostics. One
likely future benefit may permit a personalized assessment of drug
response to therapy, to help individualize drug delivery, by using
patient derived cells and blood.
V. Algorithm--Materials and Methods
[0256] A. Device Fabrication
[0257] According to one example, one or more of the microfluidic
devices described above are fabricated with Prototherm 12120 using
stereolithography (such as provided by Protolabs, Maple Plain,
Minn.). Top and bottom components of the microfluidic devices are
cast from PDMS at a 10:1 w/w base to curing agent ratio. The
components are degassed and, then, cured overnight for 4 hours at
60.degree. C. The top component contains a fluidic channel
(1.times.1 mm cross section) and ports for the top and bottom
channels. PDMS membranes, which provide a semi-permeable barrier
between the epithelium and microvascular endothelium layers, are
fabricated by casting against a DRIE-patterned silicon wafer
(50.times.50 mm). The wafer has posts that are 50 .mu.m high and
have a 7 .mu.m diameter. The posts are spaced apart at a distance
of 40 .mu.m.
[0258] To produce through-holes in the membrane using the
microfabricated post array, after pouring 100 .mu.L of PDMS onto
the wafer, a polycarbonate backing is compressed against the post
array and is baked at 60.degree. C. for 4 hrs. The membrane is
bonded to the top component using oxygen plasma treatment (e.g., 40
Watts, 800 millibars, 40 seconds; Plasma Nano, Diener Electronic,
Ebhausen, Germany), followed by bonding the assembly of the top
component and membrane to the bottom component containing an
endothelial channel (1 mm wide.times.0.2 mm high). The microfluidic
devices are sterilized using oxygen plasma treatment (100 Watts, 15
standard cubic centimeters, 30-60 seconds; PlasmaEtcher PE-100,
Plasma Etch, Reno, Nev.).
[0259] B. Cell Culture and Stimulation
[0260] After plasma treatment of the microfluidic devices, the two
chambers are pre-treated with 10% (3-aminopropyl)-trimethoxysilane
(APTMES, e.g., from Sigma) in 100% anhydrous alcohol (e.g., from
Sigma) for 10-20 minutes. The chambers are flushed sequentially
with 70% ethanol in water and 100% ethanol, and then dried at
60-80.degree. C. for two hours. Then, a mixture of rat tail
collagen I (100 .mu.g ml.sup.-1 in PBS, from BD Biosciences) and
fibronectin (e.g., 30 .mu.g/ml in PBSm, from BD Biosciences) is
introduced in both chambers of the microfluidic device. The
microfluidic device is incubated at 37.degree. C. for at least 2
hours before flushing with PBS or EGM-2 cell culture media.
[0261] Some of the microfluidic devices are used with collagen
coating alone (without cells). In other devices, HUVECs (e.g. from
Lonza) are cultured in Endothelial Growth Medium-2 (EGM-2, from
Lonza) and used between certain passages. The cells
(5-10.times.10.sup.6 cells/ml) are introduced into the
collagen-coated channels and incubated for 20 minutes at 37.degree.
C. to promote cell attachment before a second similar HUVEC
suspension is, then, introduced. The microfluidic devices are
incubated upside down for an additional 20 minutes to seed the
cells on the ceiling and walls of the lower chamber.
[0262] The lower chamber is then flushed with EGM-2 and, then, a
suspension of primary alveolar epithelial cells (e.g., from
ScienCell Research Labs, Carlsbad, Calif.; 5-6.times.10.sup.6 cells
ml.sup.-1) is introduced into the top chamber of the device. After
a few hours, the top and bottom chambers are flushed with their
respective media, and the microfluidic devices are incubated at
37.degree. C. under 5% CO.sub.2 for 3 days (with media being
replaced each day once). On day 4, the bottom vascular chamber is
set on perfusion of EGM-2 media (e.g., 30 .mu.L hr.sup.-1; 0.5%
fetal bovine serum) to provide shear to the HUVEC vessel chamber
and a continuous supply of fresh media.
[0263] On day 6, the epithelial cell media in the top chamber is
aspirated to create the air-liquid interface of the alveoli.
Hereon, the cell culture continues for another 6-8 days, after
which, in some cases, the top chamber is supplemented with 0, 5, or
100 ng ml.sup.-1 TNF-.alpha. in PBS (e.g., from Sigma) overnight or
LPS (e.g., 100 ng ml.sup.-1 of E. Coli, from Sigma,) for 2 hours to
cause cell activation. The LPS is sonicated in ultrasonic bath for
about 20-30 minutes before introducing into the microfluidic
device. After stimulation, the top chamber is clamped, the bottom
chamber is rinsed with culture media, a reservoir cut from a 3 ml
slip-tip syringe (e.g., from BD) is inserted on one end of the
lower chamber, and a 1/16-inch male luer connector (e.g., from
Qosina Corp) is inserted on the other end of the lower chamber.
[0264] C. Blood Samples and Flow Conditions
[0265] Citrated human blood (e.g., from Research Blood Components,
Cambridge, Mass.) is used within 5 hours of blood draw, to minimize
pre-analytical effects on platelet function. Platelets are labeled
with human CD41-PE antibody (e.g., 10 .mu.l ml.sup.-1, Invitrogen)
that is directly added to the blood and is incubated at room
temperature for about 10 minutes.
[0266] When analyzing the formation of fibrin, blood samples re
added with 15 .mu.g ml.sup.-1 of fluorescently labeled fibrinogen
(e.g., Alexa 488 from Invitrogen). The citrated blood is
recalcified 2 minutes after the beginning of each experiment by
adding 100 .mu.l mL.sup.-1 of a solution containing 100 mM calcium
chloride and 75 mM magnesium chloride to the blood to permit
calcium-dependent and magnesium-dependent platelet functions.
Citrated human blood (e.g., 1.2 mL) is pipetted into a fluid
reservoir fitted to one end of a microchannel on one side of the
microfluidic device.
[0267] A piece of medical grade tubing (e.g., 1.58 mm inner
diameter of Tygon S-50-HL from Saint Gobain Plastics) is fitted to
the outlet port of the device via a barbed luer lock connector
(e.g., Harvard Apparatus). The other end of the tube is connected
to a 3 ml syringe (e.g., from Becton Dickinson) through which blood
is withdrawn from the device by pulling the blood using a syringe
pump (e.g., PHD Ultra CP, Harvard Apparatus), thereby driving blood
flow through the microfluidic device. The flow rate is adjusted to
result in a wall shear rate of 250 sec.sup.-1 (e.g., approximately
10 dynes/cm.sup.2 stress). For studying platelet-endothelial
dynamics at a higher wall shear rate of 750 sec.sup.-1 (e.g.,
approximately 30 dynes/cm.sup.2 stress), a microfluidic device has
an endothelial chamber smaller in size (e.g., 0.4 mm wide.times.0.1
mm high) to facilitate experiments in which less than 1 ml of blood
is used.
[0268] D. Image Acquisition and Analysis
[0269] Platelet dynamics are visualized using time-lapse
fluorescence imaging (e.g., LD Plan Neofluar 10.times., NA 0.4;
Zeiss Axio Observer; Hamamatsu ORCA C11440 CMOS digital camera)
using an exposure time of 200 ms. Images are tiled to create a
composite panoramic view (e.g., 18,600 pixels long and 2,050 pixels
wide; 1 pixel=0.325 .mu.m). Images are archived as OME-TIFF format
files, and an image analysis is performed using, for example, Zeiss
Zen 2012 imaging software and MATLAB 2014 routines.
[0270] E. Platelet-Endothelial Dynamics Algorithm
[0271] Referring generally to FIGS. 43-50, illustrations show the
visualization and analysis of platelet-endothelial temporal
dynamics. Specifically, the illustrations include space-time
kymographs that are plotted to illustrate acquired image
time-series and graphed fluorescence at a representative pixel over
time, after removing the (linear) trend due to the increasing
platelet adhesion alone.
[0272] Referring specifically to FIGS. 43-46, representative
kymographs of a small section of a microchannel show an attachment
and detachment pattern of platelets on a collagen surface (FIG. 43)
or vessel that is untreated (FIG. 44) or TNF-.alpha. treated (FIG.
45). Referring specifically to FIG. 46, the coefficient of variance
(CV) of a fluorescence signal is observed over time at a
representative single pixel location of an image time-series of
platelet accumulation, as plotted in the kymographs shown in FIGS.
43-45.
[0273] No significant fluctuations are shown on both the
collagen-coated microfluidic surface (FIG. 43) and the surface
lined with a healthy live endothelium (FIG. 44). Thus, on collagen,
while there is platelet accumulation over time, there is hardly any
detachment or dynamical surface interactions present. A healthy
endothelium is devoid of any interaction at all. However,
TNF-.alpha. stimulated endothelium results in significant
fluctuations (FIG. 45), which is representative of occasional
attachment and detachment of platelets (dynamics).
[0274] To define a parameter that quantitates these differences at
a pixel level, a dimensionless statistical measure of dispersion is
selected as a coefficient of variance (CV). The CV is defined as a
ratio of standard deviation and mean. Although the CV at a
representative pixel is near zero for collagen and untreated
endothelium, as FIG. 46 illustrates the CV is much higher for a
treated endothelium.
[0275] Referring specifically to FIGS. 47-50, a visualization and
analysis of platelet-endothelial spatial dynamics is illustrated.
Top images are representative CV-colormaps of a large section of
the vessel where each pixel in the map represents the temporal
platelet dynamics (CV) on a collagen surface or vessel (untreated
vs TNF-.alpha. treated, with a scale bar: 100 .mu.m). Bottom graphs
show CV across the length of the channel at representative width
location for collagen (FIG. 47), untreated vessel (FIG. 48), and
vessel treated with TNF-.alpha. (FIG. 49). The dotted lines are
drawn at the 95.sup.th percentile and 5.sup.th percentile value of
the CV respectively. The graph of FIG. 50 illustrates the
interpercentile range (95.sup.th-5.sup.th percentile value) of the
CV plotted in the graphs illustrated in FIGS. 47-49, as a measure
of depicting spatial heterogeneity in platelet accumulation.
[0276] FIGS. 47-50 are directed to a much larger area of the lumen,
creating a 2-D image/map of the CV of all the pixels of the
acquired image time-series. The resulting spatial image is
reprocessed using a color map and is analysed using an intensity
palette look-up table to contrast highly active versus dormant
areas. This image intensity transformation enables spatial
visualisation of dynamic behaviour of individual platelets, in
addition to conveying an overall pattern of thrombi dynamics.
[0277] For example, FIG. 47 shows a uniform platelet adhesion
pattern with a narrow range of variance on a cell free collagen
surface. However, in FIG. 48, when blood is flowed over a healthy
endothelium, platelets show very limited reactivity with the apical
surface and, therefore, the color spectrum is almost entirely
black. Nevertheless, in FIG. 49, the thrombi patterns on
endothelium treated TNF-.alpha. are heterogeneous and fluctuate
significantly.
[0278] Furthermore, FIG. 50 is representative of a visual analysis
of the local heterogeneity within the thrombus. A plot of the CV,
over the entire length of the microchannel, at a representative
location along the width of the channel, provides the spatial
heterogeneity or fluctuations of the dispersion parameter (CV) for
a collagen coated device or an endothelium. The interpercentile
range (e.g., the difference between the 95.sup.th percentile and
5.sup.th percentile) shows that the spatial heterogeneity also
varies between the surfaces. On collagen and a healthy endothelium,
there is very little variation between the surfaces. In contrast,
the spatial heterogeneity is very high on a stimulated endothelium.
Equation 1, described above, shows the derivation of the
quantitative readout as a combination of CV and interpercentile
range.
[0279] F. Immunostaining and Histology
[0280] Fluorescence microscopy is optionally performed on an
endothelium that is fixed with 4% formaldehyde (from Sigma) and
stained with antibodies against ICAM-1 (from Santa Cruz) and
VE-Cadherin (from Santa Cruz). The endothelium is further
counterstained with phalloidin and DAPI (from Invitrogen).
[0281] G. Parmodulin ("PM2") Drug Delivery
[0282] PM2 is optionally added to the endothelial cell or
epithelial cell culture medium at a final concentration of 30
.mu.M. The cells are exposed for about 4 hours. Then, the cells are
stimulated with LPS (100 ng ml.sup.-1) for about 2 hours. LPS also
contains PM2 (30 .mu.M). Blood is, then, perfused in the
microfluidic device. In whole blood containing PM2, the blood is
added to a final concentration of 30 .mu.M, and is incubated for
about 30 minutes before perfusion.
[0283] H. Mouse Laser Injury Model
[0284] In the above described techniques, C57BL/6J mice (about 8-12
weeks old) are used. For example, the mice are purchased from the
Jackson Laboratory (Bar Harbor, Me.). Animal care and experimental
procedures are performed in accordance with and under approval of
the Beth Israel Deaconess Medical Center ("BIDMC") Institutional
Animal Care and Use Committee.
[0285] LPS is isolated from Escherichia coli serotype 0111:B4
(e.g., from Sigma-Aldrich, St Louis, Mo.). The anti-platelet
antibody CD42b conjugated to Dylight649 is purchased, for example,
from Emfret Analytics (Eibelstadt, Germany).
[0286] A laser-induced injury model of thrombosis is optionally
used to monitor thrombosis formation in cremaster arterioles and
venules in response to intraperitoneal injection of LPS (e.g., 10
mg kg') or vehicle (e.g., physiological saline solution).
Intravital microscopy of the cremaster microcirculation is
performed, with injury to a cremaster arteriolar (e.g., 30-50 .mu.m
diameter) vessel wall being induced with a Micropoint Laser System
(e.g., from Photonics Instruments, Chicago, Ill.). The Micropoint
Laser System is focused through a microscope objective, parfocal
with the focal plane and tuned to 440 nm. Data is captured
digitally in a single fluorescence channel at 647/670 nm. Data
acquisition is initiated both prior to and following a single laser
pulse for each injury. Images are captured using a CCD camera
(e.g., from Hamamatsu) at frame rates of 1/0.2 s.sup.-1 and 1/0.5
s.sup.-1, for a total of 240 seconds. The microscope system is
controlled and images are analyzed using Slidebook (e.g., from
Intelligent Imaging Innovations, Denver, Colo.). Anti-platelet
antibodies re infused into the mice prior to vessel wall
injury.
[0287] Histology Lungs are harvested in 4% paraformaldehyde.
Following overnight incubation, lungs re transferred to 70%
ethanol. Paraffin-embedded lungs are sectioned and stained with
Hematoxylin and eosin (MTS) or Masson's Trichome stain (MTS). This
work is done by the histology and microscopy core at BIDMC.
[0288] I. Statistical Analysis
[0289] Unless otherwise specifically mentioned above or in the
drawings, all data is presented as mean.+-.standard error of the
mean (SEM). Two-tailed P values are obtained from the statistical
t-test or analysis of variance (ANOVA) to compare the means. Data
analysis is optionally performed using Graphpad Prism V6.
VI. Other Embodiments
[0290] A. Organ-On-Chip (OOC) Device
[0291] Referring to FIGS. 51 and 52, a microfluidic system 700 is
configured to function in accordance with one or more of the
above-described techniques. According to the illustrated example,
the microfluidic system 700 includes an organ-on-chip ("OOC")
device 710. The OOC device 710 that includes a body 712 that is
typically made of a polymeric material. The body 712 includes a
first fluid inlet 714a and a first fluid outlet 714b. The body 712
further includes a second fluid inlet 716a and a second fluid
outlet 716b. The first fluid inlet 714a and the first fluid outlet
714b allow fluid flow through a first microchannel 724. The second
fluid inlet 716a and the second fluid outlet 716b allow fluid flow
through a second microchannel 726.
[0292] The first microchannel 724 is separated from the second
microchannel 726 by a barrier 730. The barrier 730 may be any
suitable semi-permeable barrier that permits migration of cells,
particulates, media, proteins, and/or chemicals between the first
microchannel 724 and the second microchannel 726. For example, the
barrier 730 includes gels, layers of different tissue, arrays of
micro-pillars, membranes, combinations thereof, and the like.
Depending on the application, the barrier 730 may have openings or
pores to permit the migration of cells, particulates, media,
proteins, and/or chemicals between the first microchannel 724 and
the second microchannel 726. In some preferred embodiments, the
barrier 730 is a porous membrane that includes a cell layer 734
disposed on at least a first surface of the membrane.
[0293] Optionally or alternatively, the barrier 730 includes more
than a single cell layer 734 disposed thereon. For example, the
barrier 730 includes the cell layer 734 disposed within the first
microchannel 724, the second microchannel 726, or each of the first
and second microchannels 724, 726. Additionally or alternatively,
the barrier 730 includes a first cell layer 734 disposed within the
first microchannel 724 and a second cell layer within the second
microchannel 726. Additionally or alternatively, the barrier 730
includes a first cell layer 734 and a second cell layer disposed
within the first microchannel 724, the second microchannel 726, or
each of the first and second microchannels 724, 726. In one
embodiment of the OOC device 710, the first and second
microchannels 724, 726 generally have a length of less than about 2
cm, a height of less than 200 .mu.m, and a width of less than 400
.mu.m. More details on the OOC device 710 can be found in, for
example, U.S. Pat. No. 8,647,861, which is owned by the assignee of
the present application and is incorporated by reference in its
entirety.
[0294] Referring to FIG. 53, the barrier 730 includes pores 731,
which can have various dimensions based on the barrier 730 that is
chosen. In the illustrated example, a cell layer 734 is disposed
within the first microchannel 724 and on the first upper surface of
the barrier 730. Fluid enters the first microchannel 724 and flows
from the inlet toward the outlet of the first microchannel 724. As
the fluid flows from the inlet toward the outlet of the first
microchannel 724, contact between the fluid and the surface of the
cells 734 exerts a shear stress on the cells 734. This shear stress
deform the individual cells 734, or affect other changes in the
physical or biological properties of the cells 734.
[0295] B. Exemplary Method for Determining Thrombosis Function
[0296] In accordance with the exemplary microfluidic system 700
(FIGS. 51-53), and the above-discussed techniques, an exemplary
method is directed to determining a thrombosis function. The method
includes flowing a fluid sample 800 over a top surface of the
membrane 730, which includes a endothelial cell monolayer 734. The
method further includes stimulating the fixed endothelial cell
monolayer 734 to induce formation of a clot 802, the clot being
formed via an interaction between the fixed endothelial cell
monolayer 734 and the fluid sample 800. In response to the clot
formation, a thrombosis function is determined that is associated
with the fluid sample 800 and the fixed endothelial cell monolayer
734.
[0297] In accordance with an alternative embodiment, in reference
to the above exemplary method, the fluid sample 800 includes
platelets 804 that interact with the fixed endothelial cell
monolayer 734. In this example, the thrombosis function is a
function of platelets in the fluid sample 800.
[0298] In accordance with an alternative embodiment, in reference
to the above exemplary method, the fixed endothelial cell monolayer
is derived from one or more of (a) a fixing endothelial cell
extract, (b) endothelial cell-associated proteins that are adhered
to the surface, (c) a subject from which the fluid sample 800 is
derived, (d) a subject that is different than a subject from which
the fluid sample 800 is derived, (e) fixing an endothelial cell
monolayer 734 that has been grown on the surface for a period of
time, (f) healthy cells, and (g) diseased cells.
[0299] In accordance with an alternative embodiment, in reference
to the above exemplary method, the fluid sample 800 includes one or
more of a blood sample, a serum sample, a plasma sample, a lipid
solution, and a nutrient medium.
[0300] In accordance with an alternative embodiment, in reference
to the above exemplary method, the endothelial cell monolayer 734
is physically fixed by one or more of (a) exposing to air, (b)
washing with alcohol, acetone, or a solvent that removes water, or
lipids, (c) a chemical fixative, (d) a decellularization solvent
that stabilizes surface membrane protein configuration and
cytoskeleton of a cell, (e) drying, and (f) dehydration.
[0301] In accordance with an alternative embodiment, in reference
to the above exemplary method, the chemical fixative is selected
from a group consisting of formaldehyde, paraformaldehyde,
formalin, glutaraldehyde, mercuric chloride-based fixatives,
precipitating fixatives, and dimethyl suberimidate.
[0302] In accordance with an alternative embodiment, in reference
to the above exemplary method, the method further includes
measuring at least one of temporal and spatial interaction dynamics
of cells in the fluid sample.
[0303] In accordance with an alternative embodiment, in reference
to the above exemplary method, the cells are platelets and the
spatial interaction dynamics of the cells includes at least one of
(a) binding dynamics of the platelets to the fixed endothelial cell
monolayer and (b) binding dynamics of the platelets to each
other.
[0304] In accordance with an alternative embodiment, in reference
to the above exemplary method, the method further includes storing
the top surface of the membrane 730 at (a) room temperature for a
predetermined period of time prior to said flowing the fluid sample
800 or (b) a temperature of about 4.degree. C. or lower for a
predetermined period of time prior to the flowing of the fluid
sample 800.
[0305] In accordance with an alternative embodiment, in reference
to the above exemplary method, the flowing of the fluid sample 800
is at (a) a physiological shear rate, (b) a pathological shear
rate, or (c) at a shear rate of about 50 sec.sup.-1 to about 10,000
sec.sup.-1.
[0306] C. Exemplary Microfluidic System for Determining Thrombosis
Function
[0307] In accordance with another alternative embodiment, the
microfluidic system 700 is directed to determining a thrombosis
function and includes a compartment in the form of the first
microchannel 724. The membrane 730 has the fixed endothelial cell
monolayer 734 on the top surface of the membrane 730. As such, the
compartment 724 is configured to receive the fluid sample 800
flowing over the top surface of the membrane 730 such that cells in
the fluid sample 800 interact with the fixed endothelial cell
monolayer 734.
[0308] The microfluidic system 700 further includes a detection
module 810 that is configured to detect interaction between the
cells of the fluid sample 800 and the fixed endothelial cell
monolayer 734. Additionally, the detection module 810 is configured
to detect a function of the cells in the fluid sample 800.
[0309] In accordance with an alternative embodiment, in reference
to the above exemplary microfluidic system 700, the compartment 724
includes a membrane 730 having a top surface and a bottom surface.
The first microchannel 724 is a top microchannel that is separated
from the second microchannel 726, which is a bottom microchannel,
by the membrane 730.
[0310] In accordance with an alternative embodiment, in reference
to the above exemplary microfluidic system 700, the fluid sample
800 interact with any internal surfaces of the compartment 724 or
the top surface of the membrane 730. Alternatively or additionally,
the fluid sample 800 flows through the bottom microchannel 726 and
the fluid sample 800 interact with any internal surface of the
compartment 726 or the bottom surface of the membrane 730.
[0311] In accordance with an alternative embodiment, in reference
to the above exemplary microfluidic system 700, the top surface of
the membrane 730 includes the fixed endothelial cell monolayer 734,
and the bottom surface of the membrane 730 including adhered
tissue-specific cells 814.
[0312] In accordance with an alternative embodiment, in reference
to the above exemplary microfluidic system 700, the detection
module 812 includes an imaging system 816 configured to provide
images of interaction between the cells of the fluid sample 800 and
the fixed endothelial cell monolayer 734.
[0313] In accordance with an alternative embodiment, in reference
to the above exemplary microfluidic system 700, the imaging system
816 includes a time-lapse microscopy apparatus 818.
[0314] In accordance with an alternative embodiment, in reference
to the above exemplary microfluidic system 700, the detection
module 812 includes one or more of a wide-field holography
apparatus 820, an impedance spectroscopy apparatus 822, a flow
sensor apparatus 824, and a pressure sensor apparatus 826.
[0315] In accordance with an alternative embodiment, in reference
to the above exemplary microfluidic system 700, the cells in the
fluid sample 800 include platelets, the detection module 812 being
configured to determine a function of the platelets in the fluid
sample 800.
[0316] D. Exemplary System and Method for Quantifying
Thrombosis
[0317] In accordance with another alternative embodiment, the
microfluidic system 700 is a system for quantifying thrombosis in
vitro based on physiological conditions. By way of example, the
membrane 730 is in the form of a solid substrate having a top
surface with the fixed endothelial cell monolayer 734. The
microfluidic system 700 includes the detection module 812, which is
configured to receive the solid substrate 730 and to detect spatial
and temporal interaction between cells in the fluid sample 800 and
the surface of the solid substrate 730 when the fluid sample 800 is
flowed over the surface along a flow axis F.
[0318] The system 700 includes one or more controllers 830 that are
configured to store time-lapse data of detectable signals collected
from the detection module 812, the detectable signals representing
spatial and temporal interaction between the cells of the fluid
sample 800 and the surface of the fixed endothelial cell monolayer
734. The controllers 830 generate a kymograph from at least a
portion of the stored time-lapse data, wherein a time axis of the
kymograph indicates at least a portion of the time-lapse duration,
a space axis of the kymograph indicating the detectable signals
along the flow axis.
[0319] Based on the generated kymograph, the controllers 830
determine a rate of fluctuation in a coefficient of variation (CV)
of the detectable signals to generate a temporal cell dynamics
index. The controllers 830 further determine either (i) the
presence of reactive cells in the fluid sample 800 when the
temporal cell dynamics index is higher than a temporal control
value, or (ii) the absence of reactive cells in the fluid sample
800 when the temporal cell dynamics index is no more than the
temporal control value
[0320] The system 700 further includes a display module 840 for
displaying content that is based in part on output determined by
the one or more controllers 830, wherein the content includes a
signal indicative of either presence or absence of at least one of
reactive cells or cell aggregation in the fluid sample 800.
[0321] In accordance with an alternative embodiment, in reference
to the above exemplary system 700, the fluid sample 800 is blood
and the cells are platelets.
[0322] In accordance with an alternative embodiment, in reference
to the above exemplary system 700, the detectable signals are
averaged across a width of the surface prior to generating the
kymograph.
[0323] In accordance with an alternative embodiment, in reference
to the above exemplary system 700, the width of the surface is
transverse to the flow axis.
[0324] In accordance with an alternative embodiment, in reference
to the above exemplary system 700, the controllers 830 are further
configured to generate, from at least a portion of the stored
time-lapse data, a spatial map of temporal variances of the
detectable signals, each pixel of the spatial map corresponding to
a time-averaged CV of the detectable signals.
[0325] In accordance with an alternative embodiment, in reference
to the above exemplary system 700, the controllers 830 are further
configured to determine, based on the generated spatial map, an
inter-quartile range (IQR) of the map to generate a spatial cell
dynamics index.
[0326] In accordance with an alternative embodiment, in reference
to the above exemplary system 700, the controllers 830 are further
configured to determine the presence of cell aggregation in the
fluid sample 800 when the spatial platelet dynamics index is higher
than a spatial control value, and the absence of cell aggregation
in the fluid sample 800 when the spatial platelet dynamics index is
no more than the spatial control value.
[0327] In accordance with an alternative embodiment, in reference
to the above exemplary system 700, the time-lapse data is presented
in the form of images.
[0328] In accordance with an alternative embodiment, in reference
to the above exemplary system 700, the controllers 830 are further
configured to determine cell reactivity based on a linear or
non-linear function that includes spatial and temporal dynamic
parameters.
[0329] In accordance with an alternative embodiment, a method is
directed to quantifying thrombosis in vitro based on physiological
conditions in accordance with the above exemplary system 700.
VII. Exemplary Embodiments
A. Displayed Content of Display Module
[0330] In some embodiments, referring to FIG. 62, depending on the
nature of the fluid samples and/or applications of the systems as
desired by users, the display module 908, 1108 can further display
additional content. In some embodiments where the fluid sample is
collected or derived from a subject for diagnostic assessment, the
content displayed on the display module 908, 1108 can further
comprise a signal indicative of a diagnosis of a condition (e.g.,
disease or disorder) or a state of the condition (e.g., disease or
disorder) in the subject. For example, in some embodiments where
the subject is diagnosed for platelet dysfunction, the content can
further comprise a signal indicative of a disease or disorder
induced by platelet dysfunction. Examples of the disease or
disorder induced by platelet dysfunction can include, but are not
limited to thrombosis, an inflammatory vascular disease (e.g.,
sepsis, or rheumatoid arthritis), a cardiovascular disorder (e.g.,
acute coronary syndromes, stroke, or diabetes mellitus),
vasculopathies (e.g., malaria, disseminated intravascular
coagulation), or a combination of two or more thereof.
[0331] In some embodiments wherein the fluid sample is collected or
derived from a subject for selection and/or evaluation of a
treatment regimen for a subject, the content can further comprise a
signal indicative of a treatment regimen personalized to the
subject, based on the computed temporal cell (e.g., platelet)
dynamic index and/or spatial cell (e.g., platelet) dynamic index,
as compared to a corresponding control value (e.g., based on
healthy subjects, or from the same subject before the onset of the
treatment regimen, or at an earlier time point of the treatment
regimen).
[0332] The methods and/or systems described herein can provide
tools to diagnose a disease or disorder induced by cell dysfunction
and/or abnormal cell-cell interaction in a subject. Accordingly,
another aspect described herein relates to a method of determining
if a subject is at risk, or has, a disease or disorder induced by
cell dysfunction or abnormal cell-cell interaction. The method
comprises: (a) flowing a fluid sample of the subject over a surface
comprising a fixed cell monolayer thereon; (b) detecting
interaction of cells in the fluid sample between each other and/or
with the fixed cell monolayer; and (d) identifying the subject to
be at risk, or have the disease or disorder induced by cell
dysfunction when the cell-cell interaction is higher than a
control; or identifying the subject to be less likely to have a
disease or disorder induced by cell dysfunction when the cell-cell
interaction is no more than the control.
[0333] In some embodiments, the fixed cell monolayer used in the
methods described herein can be subject-specific.
[0334] In some embodiments, the method of determining if a subject
is at risk, or has a disease or disorder induced by cell
dysfunction and/or abnormal cell-cell interaction can be used for
diagnosis and/or prognosis of a disease or disorder induced by
blood cell dysfunction (e.g., platelet dysfunction), and/or guiding
and/or monitoring of an anti-platelet and/or anti-inflammation
therapy. Accordingly, in some embodiments, the fixed endothelial
cell monolayer can comprise a fixed endothelial cell monolayer. The
fixed endothelial cell monolayer can be subject-specific. In some
embodiments, the fluid sample can comprise blood cells such as
platelets. Thus, a method of determining if a subject is at risk,
or has a disease or disorder induced by blood cell dysfunction
(e.g., platelet dysfunction) is also described herein. Non-limiting
examples of the disease or disorder induced by blood cell
dysfunction (e.g., platelet dysfunction) include, but are not
limited to thrombosis, an inflammatory vascular disease (e.g.,
sepsis, or rheumatoid arthritis), a cardiovascular disorder (e.g.,
acute coronary syndromes, stroke, or diabetes mellitus),
vasculopathies (e.g., malaria, disseminated intravascular
coagulation), or a combination of two or more thereof. In these
embodiments, the method can further comprising administering to the
subject identified to at risk or has the disease or disorder
induced by blood cell dysfunction (e.g., platelet dysfunction) an
appropriate treatment (e.g., anti-platelet therapy, or an
anti-inflammation therapy).
B. Compositions for Determining Cell-Cell Interaction
[0335] Compositions for determining cell-cell interaction are also
described herein. In one aspect, the composition comprises (a) a
solid substrate having a surface comprising a monolayer of cells of
a first type thereon; and (b) a fluid sample in contact with the
surface, wherein the fluid sample comprises cells of a second
type.
[0336] In some embodiments, the monolayer of cells of the first
type can comprise a fixed endothelial cell monolayer. In some
embodiments, the cells of the second type in the fluid sample can
comprise blood cells such as platelets.
[0337] In some embodiments, the fluid sample can comprise a blood
sample.
[0338] The cell monolayer can comprise fixed cells (e.g., fixed
endothelial cells), fixed cell extract(s) (e.g., fixed endothelial
cell extract(s)), and/or fixed cell-associated proteins (e.g.,
fixed endothelial cell-associated proteins) that are adhered to the
surface.
[0339] In some embodiments, the cell monolayer (e.g., endothelial
cell monolayer) can be derived from fixing a cell layer (e.g., an
endothelial cell monolayer) that has been grown on the surface for
a period of time, e.g., until the cell layer reaches
confluence.
[0340] The surface with which the fluid sample is in contact can be
a surface of any fluid-flowing conduit disposed in a solid
substrate. The solid substrate can be any solid substrate that is
compatible to the fluid sample and the cell monolayer. Non-limiting
examples of the solid substrate include a cell culture device, a
microscopic slide, a cell culture dish, a microfluidic device, a
microwell, and any combinations thereof.
[0341] In one embodiment, the surface can be a wall surface of a
microchannel. In one embodiment, the surface can be a surface of a
membrane. In some embodiments where the surface is a surface of a
membrane, the membrane can be configured to separate a first
chamber (e.g., a first microchannel) and a second chamber (e.g., a
second microchannel) in a microfluidic device.
[0342] In some embodiments, the microfluidic device can be
configured to comprise an organ-on-a-chip device as described
herein. An exemplary organ-on-chip can comprise a first chamber
(e.g., a first microchannel), a second chamber (e.g., a second
microchannel), and a membrane separating the first chamber and the
second chamber. In these embodiments, a first surface of the
membrane facing the first chamber can comprise the cell monolayer
(e.g., endothelial cell monolayer) thereon, and a second surface of
the membrane facing the second chamber can comprise tissue-specific
cells adhered thereon.
C. Additional Example of Applications of the Methods, Systems, and
Compositions Described Herein
[0343] The methods, systems, and compositions of various aspects
described herein can be used to determine cell-cell interaction,
e.g., but not limited to spatial and/or temporal dynamics of cells
of a first type interacting with each other or with cells of a
second type. In some embodiments, the methods, systems, and
compositions of various aspects described herein can be used to
determine blood cell dynamics (e.g., platelet dynamics).
[0344] For example, in some embodiments, the methods, systems,
and/or compositions described herein can be configured to permit a
blood cell-comprising fluid sample (e.g., platelet-comprising fluid
sample) flowing over a more reliable and physiologically relevant
endothelialized surface inflamed by a cytokine, thus mimicking the
in vivo endothelium-blood cell (e.g., platelet) crosstalk
environment, e.g., in a normal or diseased state. The blood cell
(e.g., platelet) dynamics (e.g., adhesion, translocation and/or
detachment) can be recorded and quantified, which is not possible
with the existing gold standard tests. As the blood cell (e.g.,
platelet) function/interaction can be reproduced even when the live
endothelial cells are fixed, the compositions with a fixed
endothelial cell monolayer described herein can be stored under
standard laboratory conditions for a period of time (e.g., days or
weeks) and still remain functional. Thus, the compositions
described herein can be operated near patients' bedside, e.g., in
clinics or hospitals, to determine blood cell (e.g., platelet)
dysfunction, e.g., for diagnosis of a disease or disorder induced
by blood cell (e.g., platelet) dysfunction.
[0345] In some embodiments, the compositions described herein can
further comprise tissue-specific cells. For example, in some
embodiments, a microfluidic device can comprise a first chamber
(e.g., a first microchannel), a second chamber (e.g., a second
microchannel), and a membrane separating the first chamber and the
second chamber, wherein a first surface of the membrane facing the
first chamber can comprise a endothelial cell monolayer thereon,
and a second surface of the membrane facing the second chamber can
comprise tissue-specific cells adhered thereon. A fluid comprising
blood cells (e.g., blood or blood substitute) can be introduced
into the first chamber such that blood cells can interact with the
endothelial cell monolayer. In some embodiments, the endothelial
monolayer can be an inflamed or diseased endothelial cell
monolayer. By incorporating luminal blood cell fluid transport
(e.g., a fluid comprising blood cells such as platelets) over a
fixed endothelial cell monolayer and live culture of tissue
specific cells, a physiologically relevant in vitro model of blood
cell-induced inflammation can be created to probe its
pathophysiology and/or to permit drug screening.
[0346] Accordingly, in one aspect, a method for modeling a blood
cell-induced disease or disorder in vitro is also described herein.
Examples of a blood cell-induced disease or disorder can include,
but are not limited to, thrombosis, an inflammatory vascular
disease (e.g., sepsis, or rheumatoid arthritis), a cardiovascular
disorder (e.g., acute coronary syndromes, stroke, or diabetes
mellitus), vasculopathies (e.g., malaria, disseminated
intravascular coagulation), or a combination of two or more
thereof. The method comprises flowing a fluid sample comprising
diseased blood cells (e.g., red blood cells, white blood cells,
and/or platelets) over a surface comprising an endothelial cell
monolayer (endothelium) in a cell or tissue culture device; and
detecting interaction between the blood cells in the fluid sample
and the endothelium, e.g., using the analytical methods and/or
systems described herein to determine dynamics of blood cells
binding to each other and/or to the endothelium. In some
embodiments, the endothelium can be a normal endothelium. In some
embodiments, the endothelium can be an inflamed endothelium.
[0347] The endothelium can comprise living endothelial cells or can
be fixed as described herein. In some embodiments where a fixed
endothelium is used, the disease state is induced in the
endothelium prior to fixation. In some embodiments, a fixed
endothelium can be used to model a disease when the disease state
is a result of components in the blood that do not act on the
endothelium.
[0348] In some embodiments, the diseased blood cells and/or
endothelial cells can be collected from a subject diagnosed with a
blood cell-induced disease or disorder.
[0349] In some embodiments, diseased blood cells and/or endothelial
cells can be differentiated from induced pluripotent stem cells
derived from patients carrying a blood cell-induced disease or
disorder. The diseased blood cells and/or endothelial cells can
then be manipulated, e.g., using genome engineering technologies
such as CRISPRs (clustered regularly interspaced short palindromic
repeats), to introduce or correct mutations present in the
cells.
[0350] In some embodiments where normal, healthy blood cells and/or
endothelial cells are used, the blood cells and/or endothelial
cells can be contacted with an agent (e.g., an
inflammation-inducing agent as described herein) that induces the
blood cells and/or endothelial cells to acquire at least one
phenotypic characteristic associated with a blood cell-induced
disease or disorder.
[0351] In another aspect, a method for assessing blood substitute
is also described herein. The method comprising flowing a blood
substitute over a surface comprising an endothelial cell monolayer
(endothelium) in a cell or tissue culture device, and detecting
interaction between the blood cells in the fluid sample and the
endothelium; and determining temporal and/or spatial dynamics of
blood substitute cells binding to each other and/or to the
endothelium using the analytical methods and/or systems described
herein.
[0352] As used herein, the term "blood substitute" is a substitute
for blood, which has the ability to transport and supply oxygen to
cells.
[0353] In a further aspect, a method for screening for agent(s) to
reduce at least one phenotypic characteristics of blood cell
dysfunction (e.g., platelet dysfunction) is also described herein.
The method comprises (a) flowing a fluid sample comprising diseased
blood cells (e.g., increased cell adhesion to an endothelium and/or
aggregation) over a surface comprising an endothelial cell
monolayer (endothelium) in a cell or tissue culture device; (b)
contacting the diseased blood cells and/or endothelium with a
library of candidate agents; and (c) detecting response of the
diseased blood cells and/or endothelium to the candidate agents to
identify agent(s) based on detection of the presence of a reduction
(e.g., by at least about 30% or more) in the phenotypic
characteristic of blood cell dysfunction (e.g., platelet
dysfunction).
[0354] In some embodiments, the endothelium can be a normal
endothelium. In some embodiments, the endothelium can be an
inflamed endothelium.
[0355] The candidate agents can be selected from the group
consisting of proteins, peptides, nucleic acids (e.g., but not
limited to, siRNA, anti-miRs, antisense oligonucleotides, and
ribozymes), small molecules, and a combination of two or more
thereof.
[0356] Effects of the candidate agents on the diseased blood cells
and/or endothelium can be determined by measuring response of the
cells and comparing the measured response with cells that are not
contacted with the candidate agents. Various methods to measure
cell response are known in the art, including, but not limited to,
cell labeling, immunostaining, optical or microscopic imaging
(e.g., immunofluorescence microscopy and/or scanning electron
microscopy), spectroscopy, gene expression analysis,
cytokine/chemokine secretion analysis, metabolite analysis,
polymerase chain reaction (PCR), immunoassays, ELISA, gene arrays,
spectroscopy, immunostaining, electrochemical detection,
polynucleotide detection, fluorescence anisotropy, fluorescence
resonance energy transfer, electron transfer, enzyme assay,
magnetism, electrical conductivity (e.g., trans-epithelial
electrical resistance (TEER)), isoelectric focusing,
chromatography, immunoprecipitation, immunoseparation, aptamer
binding, filtration, electrophoresis, use of a CCD camera, mass
spectroscopy, or any combination thereof. Detection, such as cell
detection, can be carried out using light microscopy with phase
contrast imaging and/or fluorescence microscopy based on the
characteristic size, shape and refractile characteristics of
specific cell types.
[0357] In some embodiments, a first surface of the membrane facing
the first channel comprises an endothelium adhered thereon. In some
embodiments, a second surface of the membrane facing the second
channel can comprise tissue-specific cells adhered thereon. As used
herein, the term "tissue-specific cells" refers to parenchymal
cells (e.g., epithelial cells) derived from a tissue or an organ,
including, e.g., but are not limited to, lung, brain, nerve
network, blood-brain-barrier, kidney, liver, heart, spleen,
pancreas, ovary, testis, prostate, skin, eye, ear, skeletal muscle,
colon, intestine, and esophagus. By way of example only, platelets
have been contemplated to play a central role in a variety of
inflammatory vascular diseases, such as sepsis, rheumatoid
arthritis etc. and other vasculopathies that may involve
endothelial barrier dysfunction, such as malaria, where lung or
brain are involved. Accordingly, in some embodiments, the second
surface of the membrane facing the second channel can comprise lung
cells or brain cells (e.g., astrocytes) to create an in vitro model
of malaria that incorporates blood transport and endothelial
barrier function.
D. Exemplary Fluid Sample
[0358] In accordance with various aspects described herein, a fluid
sample (processed or unprocessed) comprising target cells to be
analyzed can be subjected to the methods and systems described
herein. In some embodiments, the fluid sample can comprise a
biological fluid obtained from a subject. Exemplary biological
fluids obtained from a subject, e.g., a mammalian subject such as a
human subject or a domestic pet such as a cat or dog, can include,
but are not limited to, blood (including whole blood, plasma, cord
blood and serum), lactation products (e.g., milk), amniotic fluids,
sputum, saliva, urine, semen, cerebrospinal fluid, bronchial
aspirate, perspiration, mucus, liquefied feces, synovial fluid,
lymphatic fluid, tears, tracheal aspirate, and fractions
thereof.
[0359] In some embodiments, the biological fluid sample can
comprise a blood sample, a serum sample, a plasma sample, a lipid
solution, a nutrient medium, or a combination of two or more
thereof.
[0360] The biological fluid sample can be freshly collected from a
subject or a previously collected sample. In some embodiments, the
biological fluid sample used in the methods and/or systems
described herein can be collected from a subject no more than 24
hours, no more than 12 hours, no more than 6 hours, no more than 3
hours, no more than 2 hours, no more than 1 hour, no more than 30
mins or shorter.
[0361] In some embodiments, the biological fluid sample or any
fluid sample described herein can be treated with a chemical and/or
biological reagent described herein prior to use with the methods
and/or systems described herein. In some embodiments, at least one
of the chemical and/or biological reagents can be present in the
sample container before a fluid sample is added to the sample
container. For example, blood can be collected into a blood
collection tube such as VACUTAINER.RTM., which has already
contained heparin or citrate. Examples of the chemical and/or
biological reagents can include, without limitations, surfactants
and detergents, salts, cell lysing reagents, anticoagulants,
degradative enzymes (e.g., proteases, lipases, nucleases,
collagenases, cellulases, amylases), and solvents such as buffer
solutions.
[0362] In some embodiments, a fluid sample can comprise certain
cells isolated from a biological sample and resuspended in a
buffered solution or culture medium. For example, fractions of
blood or platelets can be isolated from a blood sample and
resuspended in a buffered solution or culture medium. As used
herein, the term "culture media" refers to a medium for maintaining
a tissue, an organism, or a cell population, or refers to a medium
for culturing a tissue, an organism, or a cell population, which
contains nutrients that maintain viability of the tissue, organism,
or cell population, and support proliferation and growth.
E. Example 1. Development of a Microfluidic Platelet Function
Assessment Interaction (.mu.PFA) Devices/Assays
[0363] In one aspect, microfluidic platelet function assessment
(.mu.PFA) devices comprising or consisting essentially of at least
one layer or multiple layers (e.g., at least two layers or more) of
microfluidic chambers, separated by one or more porous membranes,
were developed. The porous membranes can act as a biomimetic
interstitium and comprise living or fixed cells adhered thereto. In
some embodiments, at least one chamber can have a monolayer of
endothelial cells (normal or cytokine activated; live or fixed)
and/or extracellular matrix proteins, such as collagen, and the
chamber itself can be perfused with whole blood (a biomimetic blood
vessel--"vascular chamber") (FIG. 54A). The monolayer of
endothelial cells and/or extracellular matrix proteins can be
adhered to the side of the membrane facing the chamber and/or at
least one or more of the chamber fluid-contact surfaces (e.g., a
portion of the chamber fluid-contact surface or entire chamber
fluid-contact surface). In some embodiments, the vascular chamber
can comprise or consist essentially of a microchannel dimensioned
to be comparable to an arterial blood vessel. For example, in one
embodiment, the microchannel can be a rectangular microchannel
equivalent in size to a .about.125 .mu.m size arterial blood
vessel. In one embodiment, the microchannel can have a width of
about 400 .mu.m and a height of about 100 .mu.m (FIGS.
54B-54C).
[0364] In some embodiments, the .mu.PFA devices can comprise a
second chamber separating from the first chamber by a porous
membrane. The second chamber can comprise cultured cells of
interest (e.g., but not limited to astrocytes, pericytes,
hepatocytes, respiratory epithelial cells, and any combinations
thereof) that exhibit the functionality of a tissue of interest
(FIG. 54D). The entire device, therefore, can represent a
physiologically relevant, three dimensional, organ system that
permits blood flow and can enable dynamic interaction of blood
cells such as platelets with the endothelium and its impact on the
perivascular cells, maintained in the device.
[0365] In some embodiments, the vascular coating (e.g., the
monolayer of endothelial cells and/or extracellular matrix proteins
that are in contact with flowing blood) can also mimic inflammatory
endothelial conditions (e.g., endothelial dysfunction) by culturing
the cells in the presence of cytokines such as tumor necrosis
factor alpha (TNF-.alpha.) (FIG. 54E).
[0366] The .mu.PFA devices described herein can be utilized at
patients' bedside, e.g., for example, to analytically measure the
spatiotemporal dynamics of platelets in whole human blood at a
user-designated flow rate (shear stress). In some embodiments, no
more than 0.5 ml of blood is needed for each assay using the
.mu.PFA devices described herein.
[0367] This is one of the key departure and advancement from the
existing instruments and analytical devices that are currently
applied to measure platelet function and reactivity, because the
existing instruments and analytical devices do not incorporate
shear stresses, blood vessel geometries, and interactions with
inflamed endothelium or perivascular tissue.
[0368] In some embodiments, to assess platelet function using one
or more embodiments of the .mu.PFA devices described herein, human
whole blood can be drawn in standard 3.2% sodium citrate
vacutainers, e.g., by phlebotomy, and the assay can be performed
within a period of time of the blood draw. In one embodiment, the
assay can be performed within 6 hours of the blood draw. The
collected blood can be stored in a reservoir attached to an inlet
of the .mu.PFA devices described herein and introduced into the
"vascular chamber," for example, via a syringe pump, at a
designated flow rate (shear stress) (FIG. 54A). The blood can be
recalcified, e.g., using 100 mM calcium chloride and/or 75 mM
magnesium chloride solution (100 .mu.L/mL blood), after introduced
into the "vascular chamber" of the device. In some embodiments, the
blood can be recalcified about 2 minutes after the microfluidic
experiments begin.
[0369] In some embodiments, platelets in a blood sample can be
fluorescently labeled. In some embodiments, CD41 conjugated
antibody can be used to label platelets. Imaging can be automated
to acquire and stitch multiple image tiles (e.g., about 5-10 image
tiles) along the length of the chamber every certain period of time
(e.g., every 15 or 30 seconds). Image processing and analysis can
be done using any art-recognized programs, e.g., but not limited
to, Matlab, ImageJ and MSExcel.
[0370] For measurement of the spatiotemporal dynamics of platelet
interaction, mathematical algorithms that quantitate the dynamical
interactions between the platelets and the fixed endothelial cell
monolayer (e.g., cytokine stimulated fixed endothelial cell
monolayer) were developed as described in Example 1. Characteristic
spatial and temporal indices of platelet dynamics computed based on
acquired images of platelet interaction in the devices can be
patient-specific, e.g., when patient-specific blood sample and/or
patient-specific endothelial cells are used in the devices
described herein. Accordingly, in some embodiments, these spatial
and temporal indices of platelet dynamics can be used as prognostic
or diagnostic markers of platelet-related diseases and/or to help
in modulating antiplatelet therapy to prevent recurrent thrombosis
or bleeding. In some embodiments, these indices can be used as
quantitative markers of drug efficacy or toxicity, when the devices
described herein are used for drug or small molecule screening
(e.g., novel drug compounds). Thus, in some embodiments, these
indices can be used to determine drug toxicology.
F. Example 2. Exemplary Quantitative Algorithms for Computing
Platelet Adhesion Kinetics and/or Dynamics
[0371] This Example describes exemplary embodiments of mathematical
algorithms that each can used alone or in combination to quantitate
the dynamic interactions between platelets and one or more natural
and/or artificial surfaces. In some embodiments, the natural and/or
artificial surface can form a wall portion or entire wall on all
sides of the "vascular chamber" of the .mu.PFA devices as described
herein. The natural and/or artificial surface can be coated with
extracellular matrix molecules (e.g., collagen), cultured with an
endothelial cell monolayer (e.g., live or fixed cells with or
without cytokine stimulation), and/or a bare surface of the device
(e.g., PDMS).
[0372] Adhesion was almost completely suppressed on human umbilical
vein endothelial cell (HUVEC)-coated chambers or channels,
indicating that platelets are not adhering and are mimicking
transport in vivo inside a healthy blood vessel (FIG. 55). However,
TNF-.alpha. stimulated HUVECs (TNF+; TNF++) caused an increase in
platelet adhesion and the HUVECs treated with a higher
concentration of TNF-.alpha. caused a higher increase in platelet
adhesion as compared to the HUVECs treated with a lower TNF-.alpha.
concentration, indicative of an active and dynamic
endothelial-platelet crosstalk under inflammatory conditions.
However, these adhesion rates were still significantly lower when
compared to a collagen surface, which is one of the most potent
natural platelet agonist.
G. Example 3. A Fixed Endothelialized Surface as a Physiologically
Relevant Activator for Platelet Function Analysis
[0373] To increase practicality and reliability of the .mu.PFA
devices so that it can also be utilized at the patient bedside
environment, such as room temperature, variable humidity etc. and
stored for longer periods, the inventors have discovered that
platelet adhesion and dynamics can be similarly reproduced over an
endothelial cell monolayer that has been fixed, for example, with
3% formaldehyde, as compared to a live cultured layer. Without
wishing to be bound by theory, when the endothelium is fixed, it
can still conserve the expression of many procoagulatory proteins
such as vWF and tissue factor, that results in a spatially and
temporally heterogeneous surface, like in vivo or live cells (FIG.
58). This novel physiologically relevant surface, heterogeneously
presenting procoagulant molecules such as vWF and tissue factor,
can allow a more accurate and patient-specific platelet function
analysis. This surface is significantly different from a uniform
monolayer of collagen that is classically used in existing flow
chambers for analyzing platelet function and thrombosis. To
demonstrate reliability of measuring platelet function over this
fixed endothelial surface, platelet adhesion rate was measured on
fixed HUVECs (ENDO*) and kept for 1 day or 5 days. It was
determined that relative to the control (no treatment with
TNF-.alpha. prior to fixation), the adhesion rates of platelets
were significantly higher when the endothelial cells were treated
with TNF-.alpha. at 5 ng/ml physiological concentration, followed
by paraformaldehyde fixation (TNF+) (FIG. 59A). Yet, the fixed
endothelial surface was much less adhesive than fixed collagen
surface (COL*). In addition, the TPD (FIG. 59B) and SPD (FIG. 59C)
analysis as described in Example 2 showed similar trends, that is,
platelet dynamics over treated and fixed endothelium was elevated
relative to fixed collagen or untreated, fixed endothelium.
[0374] While the devices stored for 5 days at 4.degree. C. showed
increased dynamic behavior, as compared to when they were stored
for only a day, the difference relative to the controls was still
significant (FIGS. 59A-59C). Without wishing to be bound by theory,
fixation with paraformaldehyde can be partially reversible, and/or
the endothelial surfaces can have undergone morphological change
over time impacting the dynamics. As shown in FIG. 59A, longer
storage time does not appear to significantly affect total
adhesion.
H. Example 4. Platelet Analysis on an Organ-On-A-Chip Integrated
with Luminal Whole Blood Transport
[0375] Using a blood-brain-barrier-on-a-chip as an example, in one
embodiment, the .mu.PFA device can comprise a first chamber and a
second chamber, wherein the first chamber and the second chamber
are separated by a porous membrane. The first chamber can comprise
live cultured astrocytes, while the second chamber can comprise
endothelial cells. In some embodiments, at least one or all of the
walls (including the side of the membrane facing the second
chamber) can be lined with an endothelial cell monolayer. The
endothelial cells can be treated with or without a pro-inflammatory
factor. In this Example, both the astrocytes and the endothelial
cells were treated with TNF-.alpha. prior to exposure to blood
perfusion, thus creating an inflammatory
blood-brain-barrier-on-a-chip model including whole blood transport
that can be utilized, for example, for the study of thrombosis,
platelet activation, aggregation, platelet-endothelial and
platelet-epithelial crosstalk (FIG. 60). Platelets were observed to
be mainly on the walls of the endothelial compartment, while fibrin
has formed mostly in the static (no shear) astrocyte compartment
due to the reaction between blood fibrinogen and thrombin. The
endothelium shown in FIG. 60 is a living cell culture without
fixation. However, in some embodiments, the endothelium can be
fixed to create a blood-brain-barrier-on-a-chip model.
I. Example 5. A Platelet Function Assessment Microdevice for
Quantitative Analysis of Dynamic Platelet Interactions with
Endothelium Under Flow
[0376] Activation, aggregation, adhesion, translocation and
embolization of platelet-rich thrombi are finely controlled
dynamical processes that occur during hemostasis and thrombosis as
a result of vessel wall injury or vascular inflammation. Due to the
inherent complexity in the way platelets interact with the vessel
wall, it is challenging to study all aspects of platelet function
in a comprehensive, controlled and reproducible way. In one aspect,
described herein is a biomimetic microfluidic blood perfusion assay
where large-scale, spatiotemporal fluorescence imaging and
statistical algorithms are applied to measure and quantify
platelet-endothelial dynamics, independent of fluorescence
intensity. The device comprises, essentially consists of, or
consists of a set of microfluidic channels in which human umbilical
vein endothelial cells (HUVECs) are cultured with or without
various concentrations of one or more inflammatory cytokine, e.g.,
Tumor Necrosis Factor-.alpha. (TNF-.alpha.). The channels are then
perfused with human whole blood containing fluorescently labeled
platelets at a shear rate of about 750 s.sup.-1. Platelet-rich
thrombi form and dissociate on the endothelial cell surface over a
15 min time course and the dynamics of platelet aggregation and
thrombus embolization are quantified by analyzing temporal and
spatial variances in the fluorescent signal. This analysis revealed
a TNF-.alpha. dose-dependent increase in both the spatial and
temporal dispersion (heterogeneity) of interactions between living
platelets and endothelium in the device.
[0377] In contrast, these spatiotemporal dynamics were absent when
platelets interacted with healthy endothelium or a cell free,
collagen-coated surface that is commonly used to analyze platelet
activation and thrombus formation in most existing flow chambers.
The device and quantitative methods described here represent a
valuable tool for analyzing platelet-endothelial interactions under
pathophysiological conditions relevant for thrombosis research,
toxicology, drug screening, and clinical diagnostics.
[0378] Platelet hyper-reactivity plays a central role in the
etiology of various cardiovascular diseases and vascular disorders,
including acute coronary syndrome, stroke, pulmonary embolism, and
diabetes. Platelet activation also contributes to the failure of
implanted cardiovascular and extracorporeal devices, such as pumps,
arterial stents and artificial valves. The important role that
platelets play in multiple pathologies has resulted in the
development of a wide array of antiplatelet drugs over the last two
decades. For these reasons, it has become increasingly important to
measure platelet function in patients reliably and accurately in
laboratories for screening, diagnosis, and monitoring of
antiplatelet therapy, as well as for predicting thrombosis or
recurrent bleeding. Conventional clinical tests, such as light
transmission aggregometry or viscoelastic platelet function
analysis, have been indispensable in unraveling much of what is
currently know about platelet biology and its contribution to
thrombosis. However, these assays are limited in that they often do
not incorporate relevant fluid mechanics or physiological
interactions with the endothelial surface, which are key
determinants of thrombosis.
[0379] Parallel plate-flow chambers are macroscale devices that are
widely used to measure thrombus formation and platelet adhesion
kinetics; however, they require large sample volumes for analysis
and usually do not incorporate an endothelium. While previous
studies have reported application of arterial shear stresses in
microfluidic devices to show that thrombus formation, platelet
adhesion and aggregation can be visualized and measured on a
variety of prothrombogenic surfaces using small volumes of whole
blood or plasma, these microfluidic platelet function assays do not
permit analysis of the contributions of platelet interactions with
endothelium or a fixed endothelium that lead to complex dynamics in
which platelets tether, adhere, aggregate, detach, and/or
translocate in space and time, as they do in vivo.
J. Other Examples
[0380] In one aspect, described herein is a biomimetic platelet
function assessment device that permits robust and quantitative
analysis of how endothelial inflammation affects platelet dynamics
during thrombosis under flow in vitro. The device, being able to
emulate relevant features of an in vivo blood vessel, includes many
essential mediators of thrombosis upon stimulation (FIGS. 65A-65B).
Analysis shows that the integrated interplay between platelets,
thrombi, the vessel wall, blood-borne factors and flow dynamics can
be analyzed in the integrated system-level assay (FIG. 65C). This
new microfluidic method can therefore be used in a variety of
applications relevant for thrombosis research and clinical
practice.
[0381] Blood Samples.
[0382] Citrated human blood (Research Blood Components, Cambridge,
Mass.) was used within 5 hours of blood draw, to minimize
pre-analytical effects on platelet function. Platelets were labeled
with human CD41-PE antibody (10 .mu.l/ml, Invitrogen) directly
added to the blood and incubated at room temperature for 10 min.
When analyzing the formation of fibrin, blood samples were added
with 15 .mu.g/ml of fluorescently labeled fibrinogen (Alexa 488,
Invitrogen). The citrated blood was recalcified 2 minutes after the
beginning of each experiment by adding 100 .mu.l/ml of a solution
containing 100 mM calcium chloride and 75 mM magnesium chloride to
the blood.
[0383] Cell Culture.
[0384] Human umbilical vein endothelial cells (HUVECs, Lonza) were
cultured in Endothelial Growth Medium-2 (EGM-2, Lonza) and used
between passages 3 and 7. Before seeding HUVECs in the devices, the
microchannels were pre-treated with 1%
(3-aminopropyl)-trimethoxysilane in phosphate-buffered saline (PBS)
for 10 min, flushed sequentially with 70% ethanol in water and 100%
ethanol, and then incubated at 80.degree. C. for two hours before
rat tail collagen I (100 .mu.g/ml in PBS; BD Biosciences) was
introduced in the channels. Some of the devices were used with the
collagen coating alone (without cells) after incubation overnight
at 37.degree. C. and flushing of the channels with EGM-2. In other
studies for comparison, HUVECs (12.5.times.10.sup.6 cells/ml) were
introduced into the collagen-coated channels and incubated for 20
min at 37.degree. C. to promote cell attachment before a second
similar HUVEC suspension was then introduced and the devices were
incubated upside down for an additional 20 min to seed the cells on
the ceiling and walls of the microfluidic channels. Channels were
then flushed with EGM-2 and the devices were incubated at
37.degree. C. under 5% CO.sub.2 for 24 hours to promote HUVEC
monolayer formation on all exposed surfaces of the microchannel. In
some cases, the medium was supplemented with one or more
inflammatory cytokines (e.g., 5 or 100 ng/ml TNF-.alpha. (Sigma))
to activate the HUVEC monolayer. Fluorescence microscopy was
performed on endothelium that was fixed with 4% formaldehyde
(Sigma) and stained with antibodies against TF (Santa Cruz), vWF
(Abcam), VE-Cadherin (Santa Cruz), followed by counterstaining with
phalloidin and DAPI (Invitrogen).
[0385] Microfluidic Device Design.
[0386] The microfluidic platelet function assessment device was
designed to operate at an arterial shear rate of 750 sec.sup.-1 (30
dynes/cm.sup.2), for example, by maintaining the flow rate at 30
.mu.l/min using a syringe pump. The flow rate can vary with the
channel dimensions and/or fluid property to maintain substantially
similar ranges of the arterial shear rate. If lower flow rates are
used, red blood cell sedimentation can occur, whereas undesirably
large volumes of human donor blood are required if higher flow
rates are utilized. The channel dimensions were determined such
that they enable real-time optical microscopic imaging using a
moderate magnification (20.times., 0.4 N.A.) objective. For these
practical reasons, and to mimic the size of a small blood vessel,
the microdevice contains a microchannel that is 400 .mu.m wide, 100
.mu.m high, and 2 cm long (FIG. 65A); the hydraulic diameter of
this channel is equivalent to a 160 .mu.m diameter circular
arteriole. Six of these channels were fit on a standard
(75.times.25 mm) glass slide, allowing high assay throughput and
replicates on the same chip for testing assay reproducibility (FIG.
65B).
[0387] Engineering of an Endothelial Lining in the
Microchannel.
[0388] To recapitulate physiological interactions between flowing
platelets and the endothelial surface of living microvessels,
HUVECs were cultured on all four collagen-coated walls of the
rectangular channel. This led to the formation of rectangular
channel lined by a continuous, confluent endothelial monolayer, as
demonstrated by VE-Cadherin and F-actin staining (FIG. 71A). When
the living endothelium was stimulated with one or more inflammatory
cytokine, e.g., TNF-.alpha., monolayer integrity was maintained,
but ICAM-1 expression increased in a dose-dependent manner (FIG.
71B), which closely mimics endothelial activation observed during
inflammation in vivo. An increase in the endothelial expression of
prothrombogenic tissue factor (TF) and von Willebrand Factor (vWF)
is also found in a dose dependent manner (FIG. 72).
[0389] Spatiotemporal Visualization of Platelet Function in Flowing
Blood.
[0390] When the device is perfused with whole blood, thrombi that
are rich in platelets and fibrin form on the surface (FIG. 65C).
The spatiotemporal dynamics of fluorescent platelet activity during
formation of these thrombi on the endothelial surface was analyzed
by time-lapse imaging. As flow was stable for at least 15 min, all
experiments were carried out within the first 15 minutes. In some
embodiments, blood might begin to clot in the tubing connected to
the chip at later times (leading to decreased perfusion rates). It
was discovered that contrary to results obtained with surfaces
coated with thrombogenic proteins, the reactive surface of a living
activated endothelium is highly heterogeneous, as evidenced by
detection of significant spatial and temporal variability in
platelet adhesion, aggregation, translocation, and embolization
(data not shown). To analyze these dynamic changes in platelet
interactions with the surface of the endothelium, an automated
imaging program was created that creates a 10-frame panorama,
collectively covering a large (6 mm long.times.0.665 mm wide)
region of the microchannel. Image analysis was limited to the 200
.mu.m central region where shear rate gradients are minimal to
avoid potential boundary layer effects. This resulted in an
analytical volume of .about.0.12 .mu.L after image cropping, which
permits analysis of .about.24,000 platelets (mean platelet count:
200,000 per .mu.L whole blood) in a single composite view. The
temporal resolution was fixed to 30 sec (limited by the speed of
the image acquisition of the microscope), and imaging was
automatically performed for 30 time steps over 15 minutes (FIG.
66A). Analysis was focused on the 2.5-12.5 min time points of the
acquired time series, K(x,y,t), which covers the period of steady
growth (accelerating phase) of native whole blood clotting.
[0391] Platelet Aggregate Morphology.
[0392] Perfusion of blood through the microchannels consistently
resulted in formation of large and stable platelet aggregates when
the channels were coated with type I collagen without endothelial
cells (FIG. 66B), which is consistent with previous in vitro
studies modeling hemostasis induced by vascular wall injury. In
contrast, when the collagen-coated microchannel was covered with a
continuous living endothelial monolayer, very little platelet
interactions and aggregate formation were observed over the course
of the 15 min experiment, much as what is observed in blood flowing
in a healthy human blood vessel. However, when the endothelium was
pre-treated with varying doses of TNF-.alpha., platelet adhesion
and aggregation again resulted, but the morphology of the platelet
aggregates was clearly distinct from the aggregates that formed on
the collagen surface (FIG. 66B). The typical size of aggregates on
activated endothelium was visibly larger and they were more
sparsely distributed. Interestingly, the size, shape and
organization of the thrombi that formed on the activated
endothelium in this in vitro model were reminiscent of what has
been previously observed in vivo in animal models. Despite these
distinct qualitative observations under different conditions, no
quantitative parameters currently exist for the comparative
analysis of platelet-endothelial interactions in platelet function
assessment microdevice. In one aspect, described herein are methods
to quantify platelet-endothelial dynamic interactions in an in
vitro device, e.g., a platelet function assessment microdevice
described herein.
[0393] Platelet Adhesion and Aggregation.
[0394] Platelet adhesion and aggregation events that mediate
arterial thrombosis are mediated by glycoproteins and integrins
that are expressed on the surfaces of platelets and endothelial
cells. In most of the previous studies analyzing this process, the
response was measured by quantifying the percentage of the
endothelial surface that was covered with adherent platelets, with
a spatial resolution of a few hundred microns. This analysis is
typically performed by binary segmentation of the image after
setting a threshold fluorescence intensity for each image acquired.
The percentage area covered is then calculated as the ratio of the
number of labeled pixels to the size of the binary image and
plotted against time (FIGS. 73A-73B). There are two major
limitations of this method. First, this analysis relies on
variables that can alter platelet fluorescence (e.g., dye
concentration, labeling efficiency, light intensity, diffraction,
exposure time, etc.) that may vary from sample to sample, or
experiment to experiment. Another significant problem is that the
area averaging parameter does not provide any in-depth information
regarding the heterogeneity of the platelet aggregates or the
variation in spatiotemporal platelet dynamics (i.e., whether the
individual platelet-rich thrombi are adhering, aggregating,
translocating or embolizing).
[0395] To analyze the behavior of platelet aggregates and thrombus
forming on collagen or activated endothelium directly (i.e.,
independently of fluorescence intensity), the coefficient of
variance (CV)--the ratio of standard deviation to the mean--of the
fluorescent signal over time was calculated, and a t-projection of
the time series (K) was performed, resulting in a spatial map,
M(x,y), of platelet adhesion and aggregation dynamics (FIG. 67A).
The resulting spatial image can then be reprocessed using a color
map and analyzed using an intensity palette look-up table to
contrast highly active versus dormant areas (FIG. 67A). This image
intensity transformation enabled visualization of dynamic behavior
of individual platelet aggregates, in addition to conveying the
overall pattern of platelet aggregation. For example, a uniform
platelet adhesion pattern with a narrow range of temporal variance
on a cell free collagen surface was observed (FIG. 67A). When blood
was flowed over a healthy endothelium, platelets show very limited
reactivity with the apical surface and therefore, the color
spectrum was almost entirely black; however, the platelet patterns
on endothelium treated TNF-.alpha. were heterogeneous and
fluctuated in a dose-dependent manner (FIG. 67A and FIG. 74).
[0396] An Aggregation Index (AI) was developed to quantitatively
capture spatial variance in platelet-rich thrombus formation on
collagen and endothelium. AI corresponds to the statistical
inter-quartile range (IQR) (difference between the third and first
quartile of CV values) for each image M(x,y). This analysis
revealed that platelet behavior on the collagen surface was highly
reactive, but uniform, and there was negligible adhesion or
reactivity on surface of the healthy endothelium; thus both had a
low AI (FIG. 67B). Further, it was found that the AI of
platelet-rich thrombi on inflamed endothelium varied depending on
the dose of TNF-.alpha., and hence, the state of inflammation (FIG.
67B).
[0397] Thrombus Translocation and Embolization.
[0398] It was observed that in addition to platelets adhering and
aggregating into larger platelet-rich thrombi, the thrombi
themselves would sometimes also translocate or embolize over time.
This embolization of platelet-rich thrombi is important from a
clinical perspective, as embolisms sometimes may lead to fatal
complications. Time-averaged parameters analyzed in in vitro
assays, such as area fraction and AI, do not adequately capture
this process. Thus, to analyze temporal platelet and thrombotic
processes, such as platelet-rich thrombi translocation and
embolization, parametrically using this assay, the technique of
kymography was applied. In this method, the time series K(x,y,t) is
averaged across the x-axis and transformed to create a space-time
map N(y,t), such that the horizontal spatial axis is time (t) and
the vertical axis is platelet fluorescence along the length of
channel (y) (FIG. 68A). On a collagen surface, platelet adhesion on
the substrate increased at a steady rate over time and there were
no deviations or abrupt changes in fluorescence (FIG. 68A). Similar
analysis of an unstimulated (control) endothelial surface resulted
in a uniform and dark kymograph (FIG. 68A). However, the kymographs
on TNF-.alpha. treated endothelium exhibited great variation in the
fluorescent signal over time and space representing translocation
or embolization of platelet-rich thrombi, and this behavior altered
in a dose-dependent manner.
[0399] A fluorescence-independent quantitative parameter was
defined to capture this variation in pattern, or embolization index
(EI), which corresponds to the statistical coefficient of variance
(CV) of the image N(x,y). Similar to the results obtained with the
AI, surfaces coated with collagen or quiescent endothelium
exhibited a low EI, as the platelet behavior was either uniformly
highly reactive or negligible, respectively (FIG. 68B). This
analysis also showed that the EI of platelets on inflamed
endothelium increased with increasing doses of one or more
inflammatory cytokine, e.g., TNF-.alpha., and thus this parameter
was able to capture the processes of translocation and embolization
in this model of an inflamed vessel.
[0400] In one aspect, described herein is a new microdevice with
integrated analytical methods that represent a novel in vitro tool
for quantitatively assessing the dynamic functions of platelet and
thrombus interactions with living endothelium under flow. This
assay can be utilized in biomedical research or clinical settings
because of its ease-of-use, small sample size, automated analysis
and high information content. The novel analytical methods
described herein have several advantages relative to the existing
microfluidic thrombosis and platelet analysis models. First,
endothelial cells are an integrated component of the assay, which
allows one to study the interplay of endothelial dysfunction and
blood-derived factors in causing thrombosis or bleeding. This
advantage is clearly demonstrated by the finding presented herein
that TNF-.alpha. treatment produces dose-dependent effects on
several aspects of platelet dynamics when endothelium is present.
The other main advantage of the methods described herein is that it
simultaneously permits stochastic analysis of relevant parameters
of platelet dynamics at a large scale and by enabling high
resolution visualization of cellular responses at the single
platelet level. Moreover, the ability to quantify and compare
various parameters relating to platelet function (adhesion,
aggregation, translocation and embolization of platelet-rich
thrombi), while also carrying out morphological observations,
enabled clear comparison between the effects of the different
biomimetic surfaces. The working principle of this microfluidic
assay and/or methods described herein is also flexible, in that the
methods described herein can be integrated with a variety of other
biomedical assays and in vitro disease models. For example, the
methods or assays described herein can be easily combined with
fibrin analysis, by introducing fluorescent fibrinogen along with
labeled platelets (FIG. 65C). In some embodiments, the methods
and/or devices described herein can also be integrated with
organ-on-a-chip technology to study the effects of parenchymal
tissue damage, organ inflammation, and vascular (or perivascular)
tissue dysfunction on platelet dynamics and thrombosis in vitro in
a comprehensive fashion.
[0401] In addition, a standardized device can permit this assay to
be used to evaluate patient samples in clinical diagnostic
settings. This ability to assess the full spectrum of platelet
function enables a more informed risk assessment for thrombosis in
at-risk disease populations. For example, the fluorescence
microscopic analysis can be replaced with other imaging modalities,
such as wide-field holography or impedance spectroscopy.
K. Example 6. Whole Blood Platelet Analysis on a Chemically
Preserved Bioactive Endothelium Inside a Microfluidic Device
[0402] Thrombosis depends on blood interacting with an inflamed
vascular endothelium under flow, but it is impractical to
incorporate living endothelial cells in platelet function
diagnostic devices used in laboratories or at the bedside. In one
aspect, described herein is a microfluidic device lined by a
non-living, fixed, stimulated endothelium that supports formation
of platelet-rich thrombi as blood flows through its channels. The
clinical value of chemopreserved endothelialized devices is
demonstrated herein, e.g., by showing that they can be used to
monitor antiplatelet therapy in cardiac patients.
[0403] Mutual signaling between an inflamed endothelium and
activated platelets is commonly recognized as the cause of
disturbances in hemostasis, platelet aggregation and resulting
thrombotic disorders in various diseases, yet no reliable
diagnostic assays exist that can measure the effects of cross-talk
between platelets and an inflamed vessel wall. Microfluidic devices
that incorporate microchannels with a physiological relevant size
that are lined by living inflamed endothelium exposed to flowing
blood can be used to study thrombosis in vitro. However, it is not
practical to incorporate living endothelial cells in clinical
diagnostic devices given problems associated with culture
stability, robustness, standardization, storage, and shipping.
[0404] To this end, the inventors recapitulated
platelet-endothelial crosstalk by culturing human umbilical vein
endothelial cells (HUVECs) on all four walls of a type I collagen
coated rectangular channel (400.times.100 .mu.m), which led to
formation of a rectangular tube lined by a continuous, confluent
endothelial monolayer (FIGS. 69A-69B), as described in Example 5.
The monolayers were either left untreated or were treated for 18
hours with varying doses of the pro-inflammatory cytokine tumor
necrosis factor-alpha (TNF-.alpha.). After the endothelial
monolayer was formed and inflammatory treatment was complete, these
endothelium-lined devices were chemically preserved, for example,
by fixing them with 4% formaldehyde diluted in phosphate buffered
saline (PBS) for 15 minutes, at room temperature (FIGS. 69A-69B).
After fixation, the devices were rinsed three times with PBS and
then they were stored at 4.degree. C. for 24-36 hours before use.
Citrated human whole blood with fluorescently tagged platelets was
perfused through these fixed endothelium-lined microchannels at a
shear rate of 750 sec.sup.-1, using less than 500 .mu.l of blood
per assay (FIG. 69C). After 2 minutes, the blood was supplemented
with calcium (CaCl.sub.2) and magnesium (MgCl.sub.2) to initiate
physiological blood clotting, which was analyzed for 2.5-12.5 min.
Platelet accumulation was measured as the percentage area occupied
by the platelets in the central 200 .mu.m of the channel width
(FIG. 75).
[0405] Adding increasing doses of one or more inflammatory
cytokine, e.g., TNF-.alpha., to the endothelium prior to fixation
resulted in a dose-dependent increase in surface coverage of
platelet-rich thrombi (FIG. 69D). The morphology of these thrombi
was distinctly different from the platelet aggregates that formed
on collagen-coated device, which mimicked platelet aggregation and
adhesion that occurs on the surface of the living endothelium
during inflammation as in vivo. In contrast, there was virtually no
induction of platelet-rich thrombi formation on fixed quiescent
endothelium (FIG. 69D). In addition, no significant difference in
platelet accumulation was observed between a living and
chemopreserved endothelium, at all the tested doses of TNF-.alpha.,
thus showing that the synthesized cellular surface retains key
pro-thrombotic characteristics after fixation (FIG. 69D, n=4). It
was also shown that the platelet-rich thrombi that formed on the
chemopreserved endothelial surface were morphologically larger than
the aggregates that formed on a collagen surface, but similar to a
living endothelium (FIG. 76). Moreover, thrombi on this bioactive
surface were rich in fibrin, which also showed that their formation
was dependent on an active coagulation cascade (FIG. 76). This
activity was further substantiated when it was found that the
endothelium treated with a low dose (5 ng/ml) TNF-.alpha. expressed
higher levels of prothrombotic tissue factor (TF) and von
Willebrand Factor (vWF) than untreated, after fixation (FIG.
69E).
[0406] This data led to the investigation of whether an assay,
containing a fixed bioactive endothelial substrate, can be used to
detect anti-platelet drug dose effects, and to compare to a similar
sized collagen-coated microchannel using the standard LTA (light
transmission aggregometry). Thus, a concentration of 5 ng/ml of
TNF-.alpha. was selected for causing endothelial activation, which
is in the pathophyisologically relevant range. First, when an
antiplatelet GP IIb/IIIa antagonist drug, abciximab (ReoPro), was
added in the range 0-100 .mu.g/ml (clinical range .about.1-10
.mu.g/ml) to whole blood and platelet adhesion was measured, a
significant dose-dependent platelet inhibition was found between
untreated and 1 .mu.g/ml drug and between 1 .mu.g/ml and 10
.mu.g/ml drug (n=3, FIG. 70A). The difference between 10 .mu.g/ml
and 100 .mu.g/ml was insignificant, because at these high doses,
platelet inhibition was maximized. Surprisingly, the dose-dependent
effect of abciximab on surface coverage in a collagen-coated flow
chamber had poor sensitivity whereas abciximab-treated platelets
demonstrated no platelet aggregability by LTA in response to either
ADP (adenosine diphosphate) or collagen agonists (FIGS. 70B-70C).
This validated that the platelet aggregation measurement on the
chemopreserved endothelium provided a dynamic response across a
range of abciximab concentrations indicating that a chemopreserved
endothelium can be used to monitor anti-platelet regimens in
patients. This also showed that the surface conserved platelet
interactions via the GPIIb/IIIa pathway, involved in many
thrombotic and vascular processes.
[0407] Whole blood of patients who underwent angiography at a
cardiac catheterization lab in the clinic are regular users of
antiplatelet drugs, e.g., aspirin alone or both aspirin and
clopidogrel (Table 1).
TABLE-US-00001 TABLE 1 Clinical characteristics of subjects tested
for platelet aggregation Subject Aspirin Clopidogrel 7 + 0 8 + 0 9
+ + 16 + 0 18 + + 22 + 0 59 + + 61 + 0 63 + + 65 + + 84 0 0 85 + 0
106 + 0 109 + 0 113 0 0 117 + 0
[0408] Thus, it was next sought to perfuse whole blood of these
patients and to determine if the assays with a chemopreserved
endothelium can be used to determine effects of antiplatelet drugs.
In a subject population that was tested (n=11), it was found that
compared to healthy donors, patients showed a significant reduction
in platelet aggregation in the device described herein, consistent
also with LTA (FIGS. 70D-70F). Also, both healthy controls and
patients showed complete platelet inhibition over an untreated
endothelium but when the endothelium was stimulated, both groups
showed some signs of platelet rich thrombus formation, showing that
subjects may have a higher tendency to thrombosis when vascular
inflammation is present (FIG. 70D). These data also indicate that
patients on antiplatelet agents who show platelet adhesion similar
to healthy donors can be advised for monitoring as they may be at
risk for a thrombotic event. These results could not be reproduced
with the same sensitivity on a collagen, unendothelized coated flow
chamber (FIG. 70E), demonstrating that the assay described herein
is more reliable for assessing platelet reactivity in a clinical
setting.
[0409] In vitro humanized disease models of thrombosis offer
opportunities to significantly improve diagnostics and predict
patient outcome, where blood-endothelial interactions are involved.
In one aspect, described herein is a microfluidic device that
contains a layer of chemically preserved endothelial cells that are
either quiescent or pre-stimulated with TNF-.alpha. prior to
fixation. The chemically preserved endothelium retains its
respective passive or pro-thrombotic properties as if it were alive
(FIGS. 69A-69E). It is demonstrated herein that these devices can
be used to evaluate platelet aggregation and inhibition with drugs.
This technology can enhance platelet function analysis at bench or
bedside.
L. Exemplary Materials And Methods
[0410] Microfluidic Device Design and Fabrication.
[0411] Microfluidic device consisted of a microchannel, 400 .mu.m
wide, 100 .mu.m high and 2 cm long. It was designed using
AutoCAD.TM. software, master templates fabricated on Si (100)
wafers (University Wafer Corp.) in combination with soft
lithography using polydimethysiloxane (PDMS). Duffy et al. "Rapid
prototyping of microfluidic systems in poly(dimethylsiloxane).
Anal. Chem. 70, 4974-4984 (1998). Sylgard 184.TM. PDMS prepolymer
(Dow Corning) was cast on the silanized master that had the
positive relief of the channel features formed by SU8 2075
photoresist (MicroChem Corp). The PDMS was then cured at 60.degree.
C. in a convection oven for 120 minutes, peeled off the master, and
bonded to a PDMS coated glass slide after treating both with oxygen
plasma for 20 seconds.
[0412] Microfluidic Device Pre-Treatment and Coating.
[0413] The microfluidic devices were pre-treated and coated with
collagen before cell seeding. Devices were exposed to oxygen plasma
for 30 seconds, at a power of 50 Watts, using a PE-100.TM. plasma
sterilizer (Plasma Etch, Inc. NV, USA) and then treated with 1%
(3-aminopropyl)-trimethoxysilane (Sigma) in phosphate-buffered
saline, PBS, for 10 minutes. After rinsing with 70% ethanol and
100% ethanol, the devices were baked at 80.degree. C. for 2 hours.
A solution of 100 .mu.g/ml type I collagen from rat tail (Corning)
in PBS was then introduced in the channels. The devices were left
overnight at 37.degree. C. and 5% CO.sub.2, after which they were
rinsed with Endothelial Growth Medium-2, EGM-2 (Lonza).
[0414] Cell Culture and Chemical Preservation.
[0415] Human umbilical vein endothelial cells, HUVEC, (mixed donor,
Lonza) were kept in culture with EGM-2 and were trypsinized when
confluent. After centrifugation at 250 g, HUVEC were suspended at a
12.5 million cells/ml in EGM-2. The suspension was introduced into
the pre-treated and coated microchannels, after which the devices
were incubated upside down for 20 minutes. A fresh HUVEC suspension
was then introduced in the channels, after which the devices were
covered with EGM-2 and left at 37.degree. C., 5% CO.sub.2 for 8
hours to promote cell attachment and spreading on all surfaces of
the channel. After incubation, the channels were rinsed with EGM-2,
sometimes containing a freshly prepared solution of tumor necrosis
factor-alpha TNF-.alpha. (recombinant from E. coli, Sigma). After
incubating for 18 to 20 hours at 37.degree. C., 5% CO.sub.2, a 4%
formaldehyde solution (Sigma) was flushed through the channels and
the devices were incubated for 15 minutes at room temperature.
Finally, the devices were rinsed twice with EGM-2 and then placed
at 4.degree. C. The devices were used within 24-36 hours after
placing them at 4.degree. C.
[0416] Fluorescent Labeling of Platelets.
[0417] Platelets labeled with human CD41-PE antibody (10 .mu.l/ml,
Invitrogen) were directly added to the blood and incubated at room
temperature for 10 min. The citrated blood was recalcified 2
minutes after blood perfusion by adding 100 .mu.l/ml of a solution
containing 100 mM calcium chloride and 75 mM magnesium chloride to
the blood.
[0418] Light Transmission Aggregometry (LTA).
[0419] Blood from healthy donors was treated ex vivo with abciximab
or used untreated. These samples as well as clinical blood samples
from subjects were centrifuged at 290 g for 10 min (no brake
applied) to collect platelet rich plasma (PRP). To obtain a
reference solution for each sample, PRP was centrifuged at high
speed to pellet platelets (1,000 g, 10 min) and collect platelet
poor plasma (PPP). CaCl.sub.2 was added to each sample at a final
concentration of 1 mM to recalcify plasma before each run. Cifuni
et al. "CalDAG-GEFI and protein kinase C represent alternative
pathways leading to activation of integrin alphallbeta3 in
platelets." Blood 112, 1696-1703 (2008). LTA has then been
performed at 37.degree. C. under magnetic stirring using a
Chrono-Log Corporation instrument. Both platelet agonists, ADP
(adenosine diphosphate) and collagen, were purchased from
Chrono-Log Corporation and used as suggested by the manufacturer,
in the concentrations of 10 .mu.M and 2 .mu.g/ml, respectively.
[0420] Blood Perfusion.
[0421] 500 .mu.l of whole blood was pipetted into a fluid reservoir
fitted to one end of the microchannel on one side of the
microfluidic device. A piece of medical grade tubing (30.5 mm long,
1.58 mm inner diameter; Tygon S-50-HL, Saint Gobain Plastics) was
fitted to the outlet port of the device via a barbed luer lock
connector (Harvard Apparatus). The other end of the tube was
connected to a 3 ml syringe (Becton Dickinson) through which blood
was withdrawn from the device by pulling (30 .mu.l/min) using a
syringe pump (PHD Ultra CP, Harvard Apparatus), thereby driving
blood flow through the microchannels (FIG. 69A). Recalcification of
blood was performed after 2 min of operation to permit calcium- and
magnesium-dependent platelet-derived thrombus formation.
VIII. First Set of Alternative Embodiments
A. Embodiments A1-A9
Embodiment A1
[0422] A microchannel comprising one or more surfaces, the
microchannel having living endothelial cells on all of the
microchannel surfaces.
Embodiment A2
[0423] The microchannel of embodiment A1, wherein the living
endothelial cells are human umbilical vein endothelial cells.
Embodiment A3
[0424] The microchannel of embodiment A1, wherein the surfaces are
coated with at least one attachment molecule that supports adhesion
of the living endothelial cells.
Embodiment A4
[0425] The microchannel of embodiment A1, wherein the microchannel
includes a top surface, a bottom surface, a first side surface, and
a second side surface.
Embodiment A5
[0426] The microchannel of embodiment A4, wherein the bottom
surface includes a membrane.
Embodiment A6
[0427] The microchannel of embodiment A1, wherein the microchannel
is in fluid communication with an input port and an output
port.
Embodiment A7
[0428] The microchannel of embodiment A1, wherein the microchannel
has a width in the range of about 50 microns to about 1,000
microns.
Embodiment A8
[0429] The microchannel of embodiment A1, wherein the microchannel
has a height in the range of about 50 microns to about 200
microns.
Embodiment A9
[0430] The microchannel of embodiment A1, wherein the microchannel
includes a tube lined with a continuous, confluent layer of
endothelial cells.
B. Embodiments B1-B5
Embodiment B1
[0431] A device comprising: a body having a microchannel therein,
the microchannel including one or more surfaces, the microchannel
including living endothelial cells on all of the microchannel
surfaces.
Embodiment B2
[0432] The device of embodiment B1, wherein the microchannel
includes a top surface, a bottom surface, a first side surface, and
a second side surface.
Embodiment B3
[0433] The device of embodiment B2, wherein the bottom surface
includes a membrane.
Embodiment B4
[0434] The device of embodiment B3, wherein the membrane is at
least partially porous.
Embodiment B5
[0435] The device of embodiment B1, further comprising an input
port and an output port, the ports being in fluidic communication
with the microchannel.
C. Embodiments C1-C3
Embodiment C1
[0436] A system comprising: a) a microchannel having one or more
surfaces; b) living endothelial cells on all of the surfaces; and
(c) fluid moving through the microchannel.
Embodiment C2
[0437] The system of embodiment C1, wherein the fluid includes
whole blood that contacts the endothelial cells without
clotting.
Embodiment C3
[0438] The system of embodiment C1, wherein the fluid includes
platelets, the platelets being in contact with the endothelial
cells without clotting.
D. Embodiments D1-D8
Embodiment D1
[0439] A method comprising:
[0440] 1) providing [0441] a) a microchannel with one or more
surfaces, and [0442] b) living endothelial cells on all of the
surfaces; and
[0443] 2) introducing fluid into the microchannel.
Embodiment D2
[0444] The method of embodiment D1, wherein the living endothelial
cells are human umbilical vein endothelial cells.
Embodiment D3
[0445] The method of embodiment D1, wherein the fluid is selected
from a group consisting of a blood sample, a serum sample, a plasma
sample, a lipid solution, a nutrient medium, or a combination of
two or more thereof.
Embodiment D4
[0446] The method of embodiment D1, wherein the fluid includes
whole blood that contacts the endothelial cells without
clotting.
Embodiment D5
[0447] The method of embodiment D1, wherein the fluid includes
platelets, the platelets contacting the endothelial cells without
clotting.
Embodiment D6
[0448] The method of embodiment D1, further comprising, prior to
step 2), exposing the living endothelial cells to a
pro-inflammatory cytokine.
Embodiment D7
[0449] The method of embodiment D6, wherein the fluid includes
whole blood that contacts the endothelial ceils under conditions
such that a platelet-rich thrombus forms.
Embodiment D8
[0450] The method of embodiment D6, wherein the fluid includes
platelets, the platelets clotting upon contacting the endothelial
cells.
E. Embodiments E1-E8
Embodiment E1
[0451] A method comprising:
[0452] 1) providing [0453] a) a microchannel having one or more
surfaces, and [0454] b) fixed endothelial cells on all of the
surfaces; and
[0455] 2) introducing fluid into the microchannel.
Embodiment E2
[0456] The method of embodiment E1, wherein the fixed endothelial
cells are human umbilical vein endothelial cells.
Embodiment E3
[0457] The method of embodiment E1, wherein the fluid is selected
from a group consisting of a blood sample, a serum sample, a plasma
sample, a lipid solution, a nutrient medium, and a combination of
two or more thereof.
Embodiment E4
[0458] The method of embodiment E1, wherein the fluid includes
whole blood that contacts the endothelial cells without
clotting.
Embodiment E5
[0459] The method of embodiment E1, wherein the fluid includes
platelets, the platelets contacting the endothelial cells without
clotting.
Embodiment E6
[0460] The method of embodiment E1, wherein the endothelial cells
are physically fixed by at least one of drying and dehydration.
Embodiment E7
[0461] The method of embodiment E1, wherein the endothelial cells
are fixed by at least one of exposing to air, washing with alcohol,
acetone, or a solvent that removes at least one of water and
lipids.
Embodiment E8
[0462] The method of embodiment E1, wherein the endothelial cells
are fixed with a chemical fixative.
IX. Second Set of Alternative Embodiments
A. Embodiments A1-A10
Embodiment A1
[0463] A method of testing a drug, the method comprising:
a. providing a fluid sample of a subject, the fluid sample
including platelets; b. adding a drug to a portion of the fluid
sample to create a test sample; c. flowing the test sample through
a microchannel of a microfluidic device, the microchannel including
one or more surfaces having an endothelial cell monolayer thereon,
the endothelial cells being in a stimulated state; d. detecting
interaction between platelets in the test sample and the
endothelial cells; e. comparing the level of interaction of step d)
with that of a control; and f. determining whether the drug
interfered with a platelet function.
Embodiment A2
[0464] The method of embodiment A1, wherein the control includes
the fluid sample of the subject without the drug.
Embodiment A3
[0465] The method of embodiment A1, wherein the endothelial cells
are stimulated with a cytokine.
Embodiment A4
[0466] The method of embodiment A3, wherein the cytokine is
TNF-.alpha..
Embodiment A5
[0467] The method of embodiment A1, wherein the drug is an
antiplatelet GP IIb/IIIa antagonist.
Embodiment A6
[0468] The method of embodiment A1, wherein the drug is an
antibody.
Embodiment A7
[0469] The method of embodiment A1, wherein the drug is
abciximab.
Embodiment A8
[0470] The method of embodiment A1, wherein the microchannel has a
top surface, a bottom surface, a first side surface, and a second
side surface.
Embodiment A9
[0471] The method of embodiment A8, wherein the microchannel
includes living endothelial cells on all of the microchannel
surfaces.
Embodiment A10
[0472] The method of embodiment A8, wherein the microchannel
includes fixed endothelial cells on all of the microchannel
surfaces.
X. Third Set of Alternative Embodiments
A. Embodiments A1-A14
Embodiment A1
[0473] A method of determining if a subject is at risk, or has a
disease or disorder, induced by platelet dysfunction, the method
comprising:
a. flowing a fluid sample of the subject including platelets over a
surface having an endothelial cell monolayer thereon; b. detecting
interaction between platelets in the fluid sample and the
endothelial cells; c. comparing the level of interaction of step b)
with that of a control; and d. identifying the subject to be at
risk, or have the disease or disorder, induced by platelet
dysfunction when the platelet interaction is higher than the
control.
Embodiment A2
[0474] The method of embodiment A1, wherein the subject is at
increased risk for thrombosis.
Embodiment A3
[0475] The method of embodiment A2, further comprising selecting an
appropriate therapy and administering the therapy to the
subject.
Embodiment A4
[0476] The method of embodiment A3, wherein the therapy is
anti-platelet therapy.
Embodiment A5
[0477] The method of embodiment A3, wherein the therapy is
anti-inflammation therapy.
Embodiment A6
[0478] The method of embodiment A1, wherein the disease or disorder
induced by platelet dysfunction is an inflammatory vascular
disease.
Embodiment A7
[0479] The method of embodiment A1, wherein the disease or disorder
induced by platelet dysfunction is a cardiovascular disorder.
Embodiment A8
[0480] The method of embodiment A1, wherein the surface having an
endothelial cell monolayer is the surface of a microchannel of a
microfluidic device, the device including a body having a
microchannel therein.
Embodiment A9
[0481] The method of embodiment A8, wherein the microchannel
includes a top surface, a bottom surface, a first side surface, and
a second side surface.
Embodiment A10
[0482] The method of embodiment A9, wherein the microchannel
includes living endothelial cells on all of the microchannel
surfaces.
Embodiment A11
[0483] The method of embodiment A9, wherein the microchannel
includes fixed endothelial cells on all of the microchannel
surfaces.
Embodiment A12
[0484] The method of embodiment A9, wherein the bottom surface
includes a membrane.
Embodiment A13
[0485] The method of embodiment A12, wherein the membrane is at
least partially porous.
Embodiment A14
[0486] The method of embodiment A8, wherein the device further
includes an input port and an output port, the ports being in
fluidic communication with the microchannel.
B. Embodiments B1-B11
Embodiment B1
[0487] A method of determining if a subject is at risk or has a
disease or disorder induced by platelet dysfunction, the method
comprising:
[0488] a. flowing a fluid sample of the subject having platelets
through a microchannel of a microfluidic device, the microchannel
having one or more surfaces with an endothelial cell monolayer
thereon;
[0489] b. detecting interaction between platelets in the fluid
sample and the endothelial cells;
[0490] c. comparing the level of interaction of step b) with that
of a control; and
[0491] d. identifying the subject to be at risk or have the disease
or disorder induced by platelet dysfunction when the platelet
interaction is higher than the control.
Embodiment B2
[0492] The method of embodiment B1, wherein the subject is at
increased risk for thrombosis.
Embodiment B3
[0493] The method of embodiment B2, further comprising selecting an
appropriate therapy and administering the therapy to the
subject.
Embodiment B4
[0494] The method of embodiment B3, wherein the therapy is
anti-platelet therapy.
Embodiment B5
[0495] The method of embodiment B3, wherein the therapy is
anti-inflammation therapy.
Embodiment B6
[0496] The method of embodiment B1, wherein the disease or disorder
induced by platelet dysfunction is an inflammatory vascular
disease.
Embodiment B7
[0497] The method of embodiment B1, wherein the disease or disorder
induced by platelet dysfunction is cardiovascular disorder.
Embodiment B8
[0498] The method of embodiment B1, wherein the microchannel
includes a top surface, a bottom surface, a first side surface, and
a second side surface.
Embodiment B9
[0499] The method of embodiment B8, wherein the microchannel
includes living endothelial cells on all of the microchannel
surfaces.
Embodiment B10
[0500] The method of embodiment B8, wherein the microchannel
includes fixed endothelial cells on all of the microchannel
surfaces.
Embodiment B11
[0501] The method of embodiment B8, wherein the bottom surface
includes a membrane.
C. Embodiments C1-C4
Embodiment C1
[0502] A method of determining if a subject on an antiplatelet
agent is at risk for a thrombotic event, the method comprising:
[0503] a. flowing a fluid sample of the subject having platelets
through a microchannel of a microfluidic device, the microchannel
having one or more surfaces with an endothelial cell monolayer
thereon, the endothelial cells being in a stimulated state;
[0504] b. detecting interaction between platelets in the fluid
sample and the endothelial cells;
[0505] c. comparing the level of interaction of step b) with that
of a healthy control; and
[0506] d. identifying the subject to be at risk of a thrombotic
event when the platelet interaction is higher than the healthy
control.
Embodiment C2
[0507] The method of embodiment C1, wherein the platelet
interaction includes platelet adhesion.
Embodiment C3
[0508] The method of embodiment C1, wherein the antiplatelet agent
is aspirin.
Embodiment C4
[0509] The method of embodiment C1, wherein the antiplatelet agent
is Clopidogrel.
XI. Fourth Set of Alternative Embodiments
A. Embodiments A1-A40
Embodiment A1
[0510] A method of determining a platelet function, the method
comprising:
[0511] a. flowing a fluid sample over a surface having a fixed
endothelial cell monolayer thereon; and
[0512] b. in response to detecting interaction between platelets in
the fluid sample and the fixed endothelial cell monolayer,
determining a function of the platelets in the fluid sample.
Embodiment A2
[0513] The method of embodiment A1, wherein the fixed endothelial
cell monolayer is derived from at least one of (i) fixing an
endothelial cell extract and (ii) endothelial cell-associated
proteins that are adhered to the surface.
Embodiment A3
[0514] The method of embodiment A2, wherein the endothelial
cell-associated proteins include at least one of a procoagulatory
protein and an anti-coagulatory protein.
Embodiment A4
[0515] The method of embodiment A1, wherein the fluid sample is
selected from a group consisting of a blood sample, a serum sample,
a plasma sample, a lipid solution, a nutrient medium, and a
combination of two or more thereof.
Embodiment A5
[0516] The method of embodiment A1, wherein the surface is a
surface of a microchannel.
Embodiment A6
[0517] The method of embodiment A1, wherein the surface is a
surface of a membrane.
Embodiment A7
[0518] The method of embodiment A6, wherein the membrane is
configured to separate a first microchannel and a second
microchannel in a microfluidic device.
Embodiment A8
[0519] The method of embodiment A7, wherein the microfluidic device
is an organ-on-chip device.
Embodiment A9
[0520] The method of embodiment A7, wherein a first surface of the
membrane facing the first microchannel includes the fixed
endothelial cell monolayer thereon, and a second surface of the
membrane facing the second microchannel includes tissue-specific
cells adhered thereon.
Embodiment A10
[0521] The method of embodiment A1, wherein the fixed endothelial
cell monolayer is derived from fixing an endothelial cell monolayer
that has been grown on the surface for a period of time.
Embodiment A11
[0522] The method of embodiment A10, wherein the endothelial cell
monolayer is physically fixed by at least one of drying and
dehydration.
Embodiment A12
[0523] The method of embodiment A10, wherein the endothelial cell
monolayer is physically fixed by at least one of exposing to air
and washing with at least one of alcohol, acetone, and a solvent
that removes at least one of water and lipids.
Embodiment A13
[0524] The method of embodiment A1, wherein the endothelial cell
monolayer is fixed with a chemical fixative.
Embodiment A14
[0525] The method of embodiment A13, wherein the chemical fixative
is selected from the group consisting of formaldehyde,
paraformaldehyde, formalin, glutaraldehyde, mercuric chloride-based
fixatives, precipitating fixatives, dimethyl suberimidate (DMS),
Bouin's fixative, and a combination of two or more thereof, the
mercuric chloride-based fixatives including Helly and Zenker's
solution, the precipitating fixatives including at least one of
ethanol, methanol, and acetone.
Embodiment A15
[0526] The method of embodiment A10, wherein the endothelial cell
monolayer is fixed with a decellularization solvent that stabilizes
surface membrane protein configuration and a cytoskeleton of a
cell.
Embodiment A16
[0527] The method of embodiment A15, wherein the decellularization
solvent includes an aqueous solution having at least one of a
detergent and a high pH solution.
Embodiment A17
[0528] The method of embodiment A1, wherein endothelial cells of
the fixed endothelial cell monolayer are derived from a
subject.
Embodiment A18
[0529] The method of embodiment A1, wherein endothelial cells of
the fixed endothelial cell monolayer are differentiated from
subject-specific pluripotent stem cells.
Embodiment A19
[0530] The method of embodiment A1, wherein the fixed endothelial
cell monolayer is derived from healthy cells.
Embodiment A20
[0531] The method of embodiment A1, wherein the fixed endothelial
cell monolayer is derived from diseased cells.
Embodiment A21
[0532] The method of embodiment A20, wherein the diseased cells are
generated by contacting healthy endothelial cells with an
inflammation-inducing agent prior to the treatment with a
fixative.
Embodiment A22
[0533] The method of embodiment A21, wherein the
inflammation-inducing agent includes at least one of a physical
stimulus, a chemical agent, a biological agent, a molecular agent,
or a combination of two or more thereof.
Embodiment A23
[0534] The method of embodiment A20, wherein the diseased cells are
derived from a subject diagnosed with a disease.
Embodiment A24
[0535] The method of embodiment A1, further comprising, when the
fluid sample includes a blood sample, removing red blood cells from
the blood sample prior to flowing the blood sample over the
surface.
Embodiment A25
[0536] The method of embodiment A1, wherein the detecting includes
measuring at least one of temporal and spatial interaction dynamics
of the platelets in the fluid sample.
Embodiment A26
[0537] The method of embodiment A25, wherein the interaction
dynamics of the platelets includes at least one of binding dynamics
of the platelets to the fixed endothelial cell monolayer, binding
dynamics of the platelets to each other, and a combination
thereof.
Embodiment A27
[0538] The method of embodiment A1, wherein the platelets in the
blood sample are label-free.
Embodiment A28
[0539] The method of embodiment A1, wherein the platelets in the
blood sample are labeled with a detectable label.
Embodiment A29
[0540] The method of embodiment A28, wherein the detectable label
is a fluorescent label.
Embodiment A30
[0541] The method of embodiment A1, wherein the detecting is
performed by an imaging-based method.
Embodiment A31
[0542] The method of embodiment A30, wherein the imaging-based
method includes time-lapse microscopy.
Embodiment A32
[0543] The method of embodiment A1, wherein the detecting is
performed by at least one of a wide-field holography device, an
impedance spectroscopy device, a flow sensor, a pressure sensor,
and a combination of two or more thereof.
Embodiment A33
[0544] The method of embodiment A1, wherein the surface has been
stored at room temperature for a period of time prior to the
flowing of the fluid sample.
Embodiment A34
[0545] The method of embodiment A1, wherein the surface has been
stored at a temperature of about 4.degree. C. or lower for a period
of time prior to the flowing of the fluid sample.
Embodiment A35
[0546] The method of embodiment A33, wherein the period of time is
in the range of about 1 day to about 5 days.
Embodiment A36
[0547] The method of embodiment A1, wherein the fluid sample is
flowed at a physiological shear rate or a pathological shear
rate.
Embodiment A37
[0548] The method of embodiment A1, wherein the fluid sample is
flowed at a shear rate of about 50 sec.sup.-1 to about 10,000
sec.sup.-1.
Embodiment A38
[0549] The method of embodiment A1, wherein the fixed endothelial
cell monolayer and the fluid sample are derived from the same
subject.
Embodiment A39
[0550] The method of embodiment A1, wherein the fixed endothelial
cell monolayer and the fluid sample are derived from different
sources.
Embodiment A40
[0551] The method of embodiment A1, wherein the fluid sample
includes at least one of calcium ions and magnesium ions.
B. Embodiments B1-B2
Embodiment B1
[0552] A system for determining temporal dynamics of platelets in a
fluid sample, the system comprising:
[0553] a. a solid substrate having a surface with a fixed
endothelial cell monolayer thereon;
[0554] b. a detection module configured to receive the solid
substrate and to detect spatial and temporal interaction of
platelets in a fluid sample with the surface when the fluid sample
is flowed over the surface along a flow axis; and
[0555] c. a computer system for computing platelet dynamics, the
computer system including one or more processors and a memory to
store one or more programs, the one or more programs including
instructions for: [0556] i. storing time-lapse data of detectable
signals collected from the detection module, wherein the detectable
signals represent the spatial and temporal interaction of the
platelets with the surface, [0557] ii. generating a kymograph from
at least a portion of the stored time-lapse data, wherein the time
axis of the kymograph indicates at least a portion of the
time-lapse duration, and the space axis of the kymograph indicates
the detectable signals along the flow axis, [0558] iii. computing,
based on the generated kymograph, a rate of fluctuation in
coefficient of variation (CV) of the detectable signals to generate
a temporal platelet dynamics index, and [0559] iv. determining the
presence of reactive platelets in the fluid sample when the
temporal platelet dynamics index is higher than a temporal control
value, or determining the absence of reactive platelets in the
fluid sample when the temporal platelet dynamics index is no more
than the temporal control value; and
[0560] d. a display module for displaying a content based in part
on the computed output from the computer system, wherein the
content includes a signal indicative of at least one of the
presence of reactive platelets and platelet aggregation in the
fluid sample, or a signal indicative of at least one of the absence
of reactive platelets and platelet aggregation in the fluid
sample.
Embodiment B2
[0561] The system of embodiment B1, wherein the detectable signals
are averaged across the width of the surface, transverse to the
flow axis, prior to stacking to generate the kymograph.
C. Embodiments C1-C8
Embodiment C1
[0562] A system for determining spatial dynamics of platelets in a
fluid sample, the system comprising:
[0563] a. a solid substrate having a surface with a fixed
endothelial cell monolayer thereon;
[0564] b. a detection module configured to receive the solid
substrate and to detect spatial and temporal interaction of
platelets in a fluid sample with the surface when the fluid sample
is flowed over the surface along a flow axis;
[0565] c. a computer system for computing platelet dynamics, the
computer system including one or more processors and a memory to
store one or more programs, the one or more programs comprising
instructions for: [0566] i. storing time-lapse data of detectable
signals collected from the detection module, wherein the time-lapse
data represents the spatial and temporal interaction of the
platelets with the surface, [0567] ii. generating, from at least a
portion of the stored time-lapse data, a spatial map of temporal
variances of the detectable signals, wherein each pixel of the
spatial map corresponds to a time-averaged coefficient of variation
(CV) of the detectable signals, [0568] iii. computing, based on the
generated spatial map, an inter-quartile range (IQR) of the map to
generate a spatial platelet dynamics index, and [0569] iv.
determining the presence of platelet aggregation in the fluid
sample when the spatial platelet dynamics index is higher than a
spatial control value; or determining the absence of platelet
aggregation in the fluid sample when the spatial platelet dynamics
index is no more than the spatial control value; and
[0570] d. a display module for displaying a content based in part
on the computed output from the computer system, wherein the
content includes a signal indicative of at least one of the
presence of reactive platelets and platelet aggregation in the
fluid sample, or a signal indicative of at least one of the absence
of reactive platelets and platelet aggregation in the fluid
sample.
Embodiment C2
[0571] The system of embodiment C1, wherein the time-lapse data is
presented in the form of images.
Embodiment C3
[0572] The system of embodiment C2, wherein the detection module
includes an imaging-based device.
Embodiment C4
[0573] The system of embodiment C3, wherein the imaging-based
device includes a microscope or a microscope blade.
Embodiment C5
[0574] The system of embodiment C1, wherein the display module is
selected from a group consisting of a computer display, a screen, a
monitor, a physical printout, and a storage device, the content
being selected from a group consisting of an email, a text message,
a website, and stored information on the storage device.
Embodiment C6
[0575] The system of embodiment C1, wherein the one or more
programs include instructions for determining platelet reactivity
based on a linear or non-linear function having the spatial and
temporal dynamic parameters.
Embodiment C7
[0576] The system of embodiment C1, wherein the one or more
programs further include instructions for computing area-averaged
platelet adhesion over at least a portion of the surface.
Embodiment C8
[0577] The system of embodiment C1, wherein the fluid sample
includes a blood sample.
D. Embodiments D1-D7
Embodiment D1
[0578] A method of determining if a subject is at risk or has a
disease or disorder induced by platelet dysfunction, the method
comprising:
[0579] a. flowing a fluid sample of the subject over a surface with
a fixed endothelial cell monolayer thereon;
[0580] b. detecting interaction between platelets in the fluid
sample and the surface; and
[0581] c. identifying the subject to be [0582] at risk or have the
disease or disorder induced by platelet dysfunction when the
platelet interaction is higher than a control, or [0583] less
likely to have a disease or disorder induced by platelet
dysfunction when the platelet interaction is no more than the
control.
Embodiment D2
[0584] The method of embodiment D1, wherein the method is
implemented in a computer system having one or more processors and
a memory storing one or more programs for execution by the one or
more processors, the one or more programs including instructions
for:
[0585] i. generating a kymograph from at least a portion of
time-lapse data of detectable signals representing spatial and
temporal interaction of the platelets with the surface, wherein the
time axis of the kymograph indicates at least a portion of the
time-lapse duration, and the space axis of the kymograph indicates
the detectable signals along a flow axis;
[0586] ii. computing, based on the generated kymograph, a rate of
fluctuation in coefficient of variation (CV) of the detectable
signals to generate a temporal platelet dynamics index; and
[0587] iii. determining [0588] the presence of reactive platelets
in the fluid sample when the temporal platelet dynamics index is
higher than a temporal control value, thereby identifying the
subject to be at risk or have the disease or disorder induced by
platelet dysfunction, or [0589] the absence of reactive platelets
in the fluid sample when the temporal platelet dynamics index is no
more than the temporal control value, thereby identifying the
subject to be less likely to have a disease or disorder induced by
platelet dysfunction.
Embodiment D3
[0590] The method of embodiment D1, wherein the method is
implemented in a computer system having one or more processors and
a memory storing one or more programs for execution by the one or
more processors, the one or more programs including instructions
for:
[0591] i. generating, from at least a portion of time-lapse data of
detectable signals representing spatial and temporal interaction of
the platelets with the surface, a spatial map of temporal variances
of the detectable signals, wherein each pixel of the spatial map
corresponds to a time-averaged coefficient of variation (CV);
[0592] ii. computing, based on the generated spatial map, an
inter-quartile range (IQR) of the map to generate a spatial
platelet dynamics index; and
[0593] iii. determining [0594] the presence of platelet aggregation
in the fluid sample when the spatial platelet dynamics index is
higher than a spatial control value, thereby identifying the
subject to be at risk or have the disease or disorder induced by
platelet dysfunction, or [0595] the absence of platelet aggregation
in the fluid sample when the spatial platelet dynamics index is no
more than the spatial control value, thereby identifying the
subject to be less likely to have a disease or disorder induced by
platelet dysfunction.
Embodiment D4
[0596] The method of embodiment D1, wherein the fixed endothelial
cell monolayer is subject-specific.
Embodiment D5
[0597] The method of embodiment D1, wherein the disease or disorder
induced by platelet dysfunction is selected from a group consisting
of thrombosis, an inflammatory vascular disease, a cardiovascular
disorder, vasculopathies, and a combination of two or more thereof,
the inflammatory vascular disease including sepsis or rheumatoid
arthritis, the cardiovascular disorder including acute coronary
syndromes, stroke, or diabetes mellitus, the vasculopathies
including malaria or disseminated intravascular coagulation.
Embodiment D6
[0598] The method of embodiment D1, wherein the fluid sample
includes a blood sample.
[0599] Embodiment D7 The system of embodiment D1, wherein the fluid
sample includes a blood sample.
E. Embodiments E1-E10
Embodiment E1
[0600] A composition comprising:
[0601] a. a solid substrate having a surface with a fixed
endothelial cell monolayer thereon; and
[0602] b. a fluid sample having platelets in contact with the
surface.
Embodiment E2
[0603] The composition of embodiment E1, wherein the fluid sample
includes a blood sample.
Embodiment E3
[0604] The composition of embodiment E1, wherein the fixed
endothelial cell monolayer is derived from fixing an endothelial
cell monolayer that has been grown on the surface for a period of
time.
Embodiment E4
[0605] The composition of embodiment E3, wherein the endothelial
cell monolayer is derived from fixing at least one of endothelial
cell extract or endothelial cell-associated proteins that are
adhered to the surface.
Embodiment E5
[0606] The composition of embodiment E1, wherein the solid
substrate is selected from a group consisting of a microscopic
slide, a cell culture dish, a microfluidic device, a microwell, and
any combinations thereof.
Embodiment E6
[0607] The composition of embodiment E1, wherein the surface is a
surface of a microchannel.
Embodiment E7
[0608] The composition of embodiment E1, wherein the surface is a
surface of a membrane.
Embodiment E8
[0609] The composition of embodiment E7, wherein the membrane is
configured to separate a first microchannel and a second
microchannel in a microfluidic device.
Embodiment E9
[0610] The composition of embodiment E8, wherein the microfluidic
device is an organ-on-chip device.
Embodiment E10
[0611] The composition of embodiment E8, wherein a first surface of
the membrane is facing the first microchannel has the fixed
endothelial cell monolayer thereon, and a second surface of the
membrane facing the second microchannel has tissue-specific cells
adhered thereon.
[0612] Each of these embodiments and obvious variations thereof is
contemplated as falling within the spirit and scope of the claimed
invention, which is set forth in the following claims. Moreover,
the present concepts expressly include any and all combinations and
subcombinations of the preceding elements and aspects.
* * * * *