U.S. patent application number 14/099113 was filed with the patent office on 2014-04-03 for organ mimic device with microchannels and methods of use and manufacturing thereof.
This patent application is currently assigned to CHILDREN'S MEDICAL CENTER CORPORATION. The applicant listed for this patent is CHILDREN'S MEDICAL CENTER CORPORATION. Invention is credited to Dongeun HUH, Donald E. INGBER.
Application Number | 20140093905 14/099113 |
Document ID | / |
Family ID | 41551014 |
Filed Date | 2014-04-03 |
United States Patent
Application |
20140093905 |
Kind Code |
A1 |
INGBER; Donald E. ; et
al. |
April 3, 2014 |
ORGAN MIMIC DEVICE WITH MICROCHANNELS AND METHODS OF USE AND
MANUFACTURING THEREOF
Abstract
System and method includes a body having a central microchannel
separated by one or more porous membranes. The membranes are
configured to divide the central microchannel into a two or more
parallel central microchannels, wherein one or more first fluids
are applied through the first central microchannel and one or more
second fluids are applied through the second or more central
microchannels. The surfaces of each porous membrane can be coated
with cell adhesive molecules to support the attachment of cells and
promote their organization into tissues on the upper and lower
surface of the membrane. The pores may be large enough to only
permit exchange of gases and small chemicals, or to permit
migration and transchannel passage of large proteins and whole
living cells. Fluid pressure, flow and channel geometry also may be
varied to apply a desired mechanical force to one or both tissue
layers.
Inventors: |
INGBER; Donald E.; (Boston,
MA) ; HUH; Dongeun; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHILDREN'S MEDICAL CENTER CORPORATION |
Boston |
MA |
US |
|
|
Assignee: |
CHILDREN'S MEDICAL CENTER
CORPORATION
Boston
MA
|
Family ID: |
41551014 |
Appl. No.: |
14/099113 |
Filed: |
December 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13054095 |
Jun 30, 2011 |
8647861 |
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PCT/US2009/050830 |
Jul 16, 2009 |
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14099113 |
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61081080 |
Jul 16, 2008 |
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Current U.S.
Class: |
435/29 ;
435/287.1 |
Current CPC
Class: |
B01L 3/5027 20130101;
C12N 5/0623 20130101; C12N 5/061 20130101; G01N 33/5091 20130101;
C12N 5/0606 20130101; B01L 2300/0887 20130101; B01L 2400/0472
20130101; C12N 5/0647 20130101; C12N 5/0662 20130101; B01L 3/50273
20130101; C12M 23/58 20130101; C12N 5/0688 20130101; G01N 33/5005
20130101; C12N 5/0654 20130101; G01N 33/5088 20130101; C12M 25/02
20130101; C12N 5/0697 20130101; B01L 2300/0877 20130101; B01L
2300/163 20130101; B01L 2400/0481 20130101; B01L 2400/0487
20130101; C12N 5/0696 20130101; B01L 2300/0854 20130101; C12M 23/16
20130101; C12N 5/0671 20130101; B01L 2200/0663 20130101; C12M 29/10
20130101; C12M 21/08 20130101 |
Class at
Publication: |
435/29 ;
435/287.1 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
No.: NIH R01 ES016665-01A1 awarded by the National Institutes of
Health. The Government has certain rights in the invention.
Claims
1. (canceled)
2. A device for simulating a function or response of a tissue,
comprising: a first microchannel; a second microchannel; and a
membrane located at an interface region between the first
microchannel and the second microchannel, the membrane including a
first side facing toward the first microchannel and a second side
facing toward the second microchannel, the first side having cells
of a first type adhered thereto, the second side having cells of a
second type adhered thereto, the membrane permitting the migration
of at least one of cells, particulates, chemicals, molecules,
fluids, liquids, and gases from the first side of the membrane to
the second side of the membrane, the membrane being controllably
stretchable in at least one desired direction while a first fluid
is present in the first microchannel and a second fluid is present
in the second microchannel.
3. The device of claim 2, further including a body having a
moveable wall adjacent to the first microchannel and the second
microchannel, the moveable wall being coupled to the membrane and
being moveable so as to permit the membrane to be controllably
stretchable in the at least one desired direction.
4. The device of claim 3, wherein the body further includes a first
operating channel that is separated from at least one of the first
microchannel and the second microchannel by the moveable wall, the
moveable wall undergoing movement in response to a pressure
differential between the first operating channel and the at least
one of the first microchannel and the second microchannel.
5. The device of claim 2, further including a motor coupled to the
membrane to permit the membrane to be controllably stretchable.
6. The device of claim 2, further including a mechanical actuator
coupled to the membrane to permit the membrane to be controllably
stretchable.
7. The device of claim 2, wherein the first microchannel has a
different cross-sectional size than the second microchannel.
8. The device of claim 7, wherein the membrane divides a main
channel within a body of the device into the first microchannel and
the second microchannel, the membrane being vertically offset from
a center of the main channel to create the different
cross-sectional sizes.
9. The device of claim 2, further including a body defined by at
least an upper layer and a lower layer, the membrane being a
membrane layer located between the upper layer and the lower layer,
the first microchannel being at least partially defined by the
upper layer and the membrane layer, the second microchannel being
at least partially defined by the lower layer and the membrane
layer.
10. The device of claim 9, wherein the upper layer, the lower
layer, and the membrane layer are coupled together through a
bonding process.
11. The device of claim 2, further including a body defined by an
upper component and a lower component that are made by introducing
material into a master that forms the first microchannel and the
second microchannel in the upper component and the lower component,
respectively.
12. The device of claim 2, wherein the first microchannel has the
same cross-sectional size as the second microchannel.
13. The device of claim 2, wherein the first microchannel and the
second microchannel are at least partially defined by curved
walls.
14. The device of claim 2, wherein the membrane controllably
stretches in a manner so as to undergo a cyclic movement
pattern.
15. The device of claim 2, wherein the membrane controllably
stretches in a manner so as to undergo an irregular movement
pattern.
16. The device of claim 2, wherein the first fluid and the second
fluid are the same fluid.
17. The device of claim 2, wherein at least one of the first fluid
and the second fluid is a gaseous fluid.
18. The device of claim 2, wherein at least one of the first fluid
and the second fluid is a liquid.
19. A method of simulating a function or a response of a tissue in
a device having a membrane located at an interface region between a
first microchannel and a second microchannel, a first side of the
membrane being exposed to the first microchannel and having a first
type of cells adhered thereto, the method comprising: moving a
first fluid through the first microchannel; moving a second fluid
through the second microchannel; and while (i) the first fluid is
adjacent to the first type of living cells within the first
microchannel and (ii) the second fluid is within the second
microchannel, stretching the membrane in a first direction so as to
apply a force to the first type of living cells adhered to the
first side of the membrane.
20. The method of claim 19, wherein a second side of the membrane
has a second type of living cells adhered thereto, the membrane
permitting the migration of at least one of cells, particulates,
chemicals, molecules, fluids, liquids, and gases between the first
type of cells and the second type of cells.
21. The method of claim 19, wherein the stretching includes moving
a movable wall to which the membrane is coupled.
22. The method of claim 19, wherein the stretching includes using a
motor that causes the membrane to move.
23. The method of claim 19, wherein the stretching includes using a
mechanical actuator that causes the membrane to move.
24. The method of claim 9, further including, after the stretching,
allowing the membrane to retract so to relieve the force applied to
the first type of living cells.
25. The method of claim 24, further including, repeating (i) the
stretching the membrane and (ii) the allowing the membrane to
retract so as to apply repeated forces to the first type of living
cells.
26. The method of claim 25, wherein the repeated forces are applied
in a cyclical pattern.
27. The method of claim 25, wherein the repeated forces are applied
in a non-uniform pattern.
28. The method of claim 25, wherein the repeated forces are of
different durations.
29. The method of claim 19, wherein at least one of the first fluid
and the second fluid is a gaseous fluid.
30. The method of claim 19, wherein the moving the first fluid
further includes controlling a flow and content of the first fluid
within the first microchannel.
31. The method of claim 30, further including introducing an agent
into the first fluid within the first microchannel and measuring a
response of the first type of living cells to the agent.
32. The method of claim 31, wherein the measuring the response of
the first type of living cells to the agent occurs while the force
is applied.
33. The method of claim 19, further including while the first fluid
is adjacent to the first type of living cells within the first
microchannel, stretching the membrane in a second direction so as
to apply a different force to the first type of living cells
adhered to the first side of the membrane, the first direction
being different from the second direction.
34. The method of claim 19, wherein at least one of the first fluid
and the second fluid is a liquid fluid.
35. The method of claim 19, wherein the stretching occurs while the
least one of the first fluid and the second fluid is moving,
respectively, in the first microchannel and the second
microchannel.
36. The method of claim 19, wherein the first fluid and the second
fluid are the same fluid.
37. A device for simulating a function or response of a tissue,
comprising: a body having a main microchannel and a
membrane-mounting region; and a membrane extending generally within
a plane across at least a portion of the main microchannel and
separating the main microchannel into a first microchannel and a
second microchannel, the membrane being coupled to the body at the
membrane-mounting region, the membrane including a first side
facing toward the first microchannel and a second side facing
toward the second microchannel, the first side having cells of a
first type adhered thereto; wherein the membrane is controllably
stretchable and retractable in at least a first direction along the
plane while a first fluid is present in the first microchannel and
a second fluid is present in the second microchannel.
38. The device of claim 37, wherein the body includes a moveable
wall adjacent to the main microchannel, the moveable wall including
the membrane-mounting region and being coupled to the membrane, the
movable wall causing the membrane to be controllably stretched and
retracted in the first direction.
39. The device of claim 38, wherein the moveable wall separates a
first operating channel from the main microchannel, the moveable
wall undergoing movement in response to a pressure differential
between the first operating channel and the main microchannel.
40. The device of claim 37, further including a motor that allows
the membrane to be controllably stretched and retracted in the
first direction.
41. The device of claim 37, further including a mechanical actuator
that allows the membrane to be controllably stretched and retracted
in the first direction.
42. The device of claim 37, wherein the second side of the membrane
has a second type of living cells adhered thereto, the membrane
permitting the migration of at least one of cells, particulates,
chemicals, molecules, fluids and gases between the first type of
cells and the second type of cells.
43. The device of claim 37, wherein at least one of the first fluid
and the second fluid is a liquid fluid.
44. The device of claim 37, wherein at least one of the first fluid
and the second fluid is a gaseous fluid.
45. The device of claim 37, wherein the stretching and retracting
occurs while the least one of the first fluid and the second fluid
is moving, respectively, in the first microchannel and the second
microchannel.
46. The device of claim 37, wherein the first fluid and the second
fluid are the same fluid.
47. A device for simulating a function or response of a tissue,
comprising: a body having a first microchannel and a second
microchannel, the first microchannel having a first fluid therein
and the second microchannel having a second fluid therein; and a
membrane located at an interface region between the first
microchannel and the second microchannel, the membrane including a
first side facing toward the first microchannel and a second side
facing toward the second microchannel, the first side having cells
of at least a first type adhered thereto, the second side having
cells of at least a second type adhered thereto, the membrane
permitting cellular communication between the first type of cells
and the second type of cells, the membrane being controllably
stretchable while the first fluid is present in the first
microchannel and the second fluid is present in the second
microchannel.
48. The device of claim 47, further including a motor that allows
the membrane to be controllably stretched in a first direction.
49. The device of claim 47, wherein at least one of the first fluid
and the second fluid is a liquid fluid.
50. The device of claim 47, wherein the first fluid and the second
fluid are the same fluid.
51. The device of claim 47, wherein at least one of the first fluid
and the second fluid is a gaseous fluid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of co-pending
U.S. patent application Ser. No. 13/054,095 filed on Jan. 4, 2011,
which application is a 371 National Phase Entry Application of
International Application No. PCT/US2009/050830 filed Jul. 16,
2009, which designates the U.S., and which claims the benefit of
priority under 35 U.S.C. .sctn.119(e) of U.S. Provisional
Application No. 61/081,080 filed Jul. 16, 2008, the contents of
which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0003] The present disclosure relates generally to an organ mimic
device with microchannels and methods of use and manufacturing
thereof.
BACKGROUND
[0004] Mechanical forces--pushes, pulls, tensions,
compressions--are important regulators of cell development and
behavior. Tensegrity provides the structure that determines how
these physical forces are distributed inside a cell or tissue, and
how and where they exert their influence.
[0005] In the human body, most cells are constantly subjected to
mechanical forces, such as tension or compression. Application of
mechanical strain to cells in culture simulates the in vivo
environment, causing dramatic morphologic changes and biomechanical
responses in the cells. There are both long and short term changes
that occur when cells are mechanically loaded in culture, such as
alterations in the rate and amount of DNA or RNA synthesis or
degradation, protein expression and secretion, the rate of cell
division and alignment, changes in energy metabolism, changes in
rates of macromolecular synthesis or degradation, and other changes
in biochemistry and bioenergetics.
[0006] Every cell has an internal scaffolding, or cytoskeleton, a
lattice formed from molecular "struts and wires". The "wires" are a
crisscrossing network of fine cables, known as microfilaments, that
stretch from the cell membrane to the nucleus, exerting an inward
pull. Opposing the pull are microtubules, the thicker
compression-bearing "struts" of the cytoskeleton, and specialized
receptor molecules on the cell's outer membrane that anchor the
cell to the extracellular matrix, the fibrous substance that holds
groups of cells together. This balance of forces is the hallmark of
tensegrity.
[0007] Tissues are built from groups of cells, like eggs sitting on
the "egg carton" of the extracellular matrix. The receptor
molecules anchoring cells to the matrix, known as integrins,
connect the cells to the wider world. Mechanical force on a tissue
is felt first by integrins at these anchoring points, and then is
carried by the cytoskeleton to regions deep inside each cell.
Inside the cell, the force might vibrate or change the shape of a
protein molecule, triggering a biochemical reaction, or tug on a
chromosome in the nucleus, activating a gene.
[0008] Cells also can be said to have "tone," just like muscles,
because of the constant pull of the cytoskeletal filaments. Much
like a stretched violin string produces different sounds when force
is applied at different points along its length, the cell processes
chemical signals differently depending on how much it is
distorted.
[0009] A growth factor will have different effects depending on how
much the cell is stretched. Cells that are stretched and flattened,
like those in the surfaces of wounds, tend to grow and multiply,
whereas rounded cells, cramped by overly crowded conditions, switch
on a "suicide" program and die. In contrast, cells that are neither
stretched nor retracted carry on with their intended functions.
[0010] Another tenet of cellular tensegrity is that physical
location matters. When regulatory molecules float around loose
inside the cell, their activities are little affected by mechanical
forces that act on the cell as a whole. But when they're attached
to the cytoskeleton, they become part of the larger network, and
are in a position to influence cellular decision-making Many
regulatory and signaling molecules are anchored on the cytoskeleton
at the cell's surface membrane, in spots known as adhesion sites,
where integrins cluster. These prime locations are key
signal-processing centers, like nodes on a computer network, where
neighboring molecules can receive mechanical information from the
outside world and exchange signals.
[0011] Thus, assessing the full effects of drugs, drug delivery
vehicles, nanodiagnostics or therapies or environmental stressors,
such as particles, gases, and toxins, in a physiological
environment requires not only a study of the cell-cell and
cell-chemical interactions, but also a study of how these
interactions are affected by physiological mechanical forces in
both healthy tissues and tissues affected with diseases.
[0012] Methods of altering the mechanical environment or response
of cells in culture have included wounding cells by scraping a
monolayer, applying magnetic or electric fields, or by applying
static or cyclic tension or compression with a screw device,
hydraulic pressure, or weights directly to the cultured cells.
Shear stress has also been induced by subjecting the cells to fluid
flow. However, few of these procedures have allowed for
quantitation of the applied strains or provided regulation to
achieve a broad reproducible range of cyclic deformations within a
culture microenvironment that maintains physiologically relevant
tissue-tissue interactions.
[0013] Living organs are three-dimensional vascularized structures
composed of two or more closely apposed tissues that function
collectively and transport materials, cells and information across
tissue-tissue interfaces in the presence of dynamic mechanical
forces, such as fluid shear and mechanical strain. Creation of
microdevices containing living cells that recreate these
physiological tissue-tissue interfaces and permit fluid flow and
dynamic mechanical distortion would have great value for study of
complex organ functions, e.g., immune cell trafficking, nutrient
absorption, infection, oxygen and carbon dioxide exchange, etc.,
and for drug screening, toxicology, diagnostics and
therapeutics.
[0014] The alveolar-capillary unit plays a vital role in the
maintenance of normal physiological function of the lung as well as
in the pathogenesis and progression of various pulmonary diseases.
Because of the complex architecture of the lung, the small size of
lung alveoli and their surrounding microvessels, and the dynamic
mechanical motions of this organ, it is difficult to study this
structure at the microscale.
[0015] The lung has an anatomically unique structure having a
hierarchical branching network of conducting tubes that enable
convective gas transport to and from the microscopic alveolar
compartments where gas exchange occurs. The alveolus is the most
important functional unit of the lung for normal respiration, and
it is most clinically relevant in that it is the blood-gas barrier
or interface, as well as the site where surfactants act to permit
air entry and where immune cells, pathogens and fluids accumulate
in patients with acute respiratory distress syndrome (ARDS) or
infections, such as pneumonia.
[0016] The blood-gas barrier or tissue-tissue interface between the
pulmonary capillaries and the alveolar lumen is composed of a
single layer of alveolar epithelium closely juxtaposed to a single
layer of capillary endothelium separated by a thin extracellular
matrix (ECM), which forms through cellular and molecular
self-assembly in the embryo. Virtually all analysis of the function
of the alveolar-capillary unit has been carried out in whole animal
studies because it has not been possible to regenerate this
organ-level structure in vitro.
[0017] A major challenge lies in the lack of experimental tools
that can promote assembly of multi-cellular and multi-tissue
organ-like structures that exhibit the key structural organization,
physiological functions, and physiological or pathological
mechanical activity of the lung alveolar-capillary unit, which
normally undergoes repeated expansion and contraction during each
respiratory cycle. This limitation could be overcome if it were
possible to regenerate this organ-level structure and recreate its
physiological mechanical microenvironment in vitro. However, this
has not been fully accomplished.
[0018] What is needed is a organ mimic device capable of being used
in vitro or in vivo which performs tissue-tissue related functions
and which also allows cells to naturally organize in the device in
response to not only chemical but also mechanical forces and allows
the study of cell behavior through a membrane which mimics
tissue-tissue physiology.
Overview
[0019] System and method comprises a body having a central
microchannel separated by one or more porous membranes. The
membranes are configured to divide the central microchannel into a
two or more closely apposed parallel central microchannels, wherein
one or more first fluids are applied through the first central
microchannel and one or more second fluids are applied through the
second or more central microchannels. The surfaces of each porous
membrane can be coated with cell adhesive molecules to support the
attachment of cells and promote their organization into tissues on
the upper and lower surface of each membrane, thereby creating one
or more tissue-tissue interfaces separated by porous membranes
between the adjacent parallel fluid channels. The membrane may be
porous, flexible, elastic, or a combination thereof with pores
large enough to only permit exchange of gases and small chemicals,
or large enough to permit migration and transchannel passage of
large proteins, as well as whole living cells. Fluid pressure, flow
characteristics and channel geometry also may be varied to apply a
desired fluid shear stress to one or both tissue layers.
[0020] In an embodiment, operating channels adjacent to the central
channel are applied a positive or negative pressure which creates a
pressure differential that causes the membrane to selectively
expand and retract in response to the pressure, thereby further
physiologically simulating mechanical force of a living
tissue-tissue interface.
[0021] In another embodiment, three or more parallel microchannels
are separated by a plurality of parallel porous membranes which are
lined by a common tissue type in the central channel and two
different tissue types on the opposite sides of the membranes in
the two outer channels. An example would be a cancer mimic device
in which cancer cells are grown in the central microchannel and on
the inner surfaces of both porous membranes, while capillary
endothelium is grown on the opposite surface of one porous membrane
and lymphatic endothelium is grown on the opposite surface of the
second porous membrane. This recreates the tumor microarchitecture
and permits study of delivery of oxygen, nutrients, drugs and
immune cells via the vascular conduit as well as tumor cell egress
and metastasis via the lymphatic micro channel.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The accompanying drawings, which are incorporated into and
constitute a part of this specification, illustrate one or more
examples of embodiments and, together with the description of
example embodiments, serve to explain the principles and
implementations of the embodiments. In the drawings:
[0023] FIG. 1 illustrates a block diagram of a system employing an
example organ mimic device in accordance with an embodiment.
[0024] FIG. 2A illustrates a perspective view of a organ mimic
device in accordance with an embodiment.
[0025] FIG. 2B illustrates an exploded view of the organ mimic
device in accordance with an embodiment.
[0026] FIGS. 2C-2D illustrate perspective views of tissue-tissue
interface regions of the device in accordance with an
embodiment.
[0027] FIGS. 2E-2G illustrate top down cross sectional views of the
tissue-tissue interface regions of the device in accordance with
one or more embodiments.
[0028] FIGS. 3A-3B illustrate perspective views of tissue-tissue
interface regions of the device in accordance with an
embodiment.
[0029] FIGS. 3C-3E illustrate perspective views of the membrane in
accordance with one or more embodiments.
[0030] FIGS. 4A-4C illustrate perspective views of the formation of
the membrane of a two channel device in accordance with an
embodiment.
[0031] FIG. 4D illustrates a side view of the membrane of the
tissue-tissue interface device in accordance with an
embodiment.
[0032] FIGS. 5A-5E illustrate perspective views of the formation of
the organ mimic device in accordance with an embodiment.
[0033] FIG. 6 illustrates a system diagram employing an organ mimic
device with multiple channels in accordance with an embodiment.
[0034] FIGS. 7A-7B illustrate perspective views of the organ mimic
device in accordance with an embodiment.
[0035] FIG. 7C illustrates a side view of the membrane of the organ
mimic device in accordance with an embodiment.
[0036] FIGS. 8 and 9 illustrate ROS generation over time in
accordance with an experiment conducting with the present
device.
DESCRIPTION OF EXAMPLE EMBODIMENTS
[0037] Example embodiments are described herein in the context of
an organ simulating device and methods of use and manufacturing
thereof. Those of ordinary skill in the art will realize that the
following description is illustrative only and is not intended to
be in any way limiting. Other embodiments will readily suggest
themselves to such skilled persons having the benefit of this
disclosure. Reference will now be made in detail to implementations
of the example embodiments as illustrated in the accompanying
drawings. The same reference indicators will be used throughout the
drawings and the following description to refer to the same or like
items. It is understood that the phrase "an embodiment" encompasses
more than one embodiment and is thus not limited to only one
embodiment for brevity's sake.
[0038] In accordance with this disclosure, the organ mimic device
(also referred to as "present device") is preferably utilized in an
overall system incorporating sensors, computers, displays and other
computing equipment utilizing software, data components, process
steps and/or data structures. The components, process steps, and/or
data structures described herein with respect to the computer
system with which the organ mimic device is employed may be
implemented using various types of operating systems, computing
platforms, computer programs, and/or general purpose machines. In
addition, those of ordinary skill in the art will recognize that
devices of a less general purpose nature, such as hardwired
devices, field programmable gate arrays (FPGAs), application
specific integrated circuits (ASICs), or the like, may also be used
without departing from the scope and spirit of the inventive
concepts disclosed herein.
[0039] Where a method comprising a series of process steps is
implemented by a computer or a machine with use with the organ
mimic device described below and those process steps can be stored
as a series of instructions readable by the machine, they may be
stored on a tangible medium such as a computer memory device (e.g.,
ROM (Read Only Memory), PROM (Programmable Read Only Memory),
EEPROM (Electrically Erasable Programmable Read Only Memory), FLASH
Memory, Jump Drive, and the like), magnetic storage medium (e.g.,
tape, magnetic disk drive, and the like), optical storage medium
(e.g., CD-ROM, DVD-ROM, paper card, paper tape and the like) and
other types of program memory.
[0040] Embodiments of the present device can be applied in numerous
fields including basic biological science, life science research,
drug discovery and development, drug safety testing, chemical and
biological assays, as well as tissue and organ engineering. In an
embodiment, the organ mimic device can be used as microvascular
network structures for basic research in cardiovascular, cancer,
and organ-specific disease biology. Furthermore, one or more
embodiments of the device find application in organ assist devices
for liver, kidney, lung, intestine, bone marrow, and other organs
and tissues, as well as in organ replacement structures.
[0041] The cellular responses to the various environmental cues can
be monitored using various systems that can be combined with the
present device. One can monitor changes in pH using well known
sensors. One can also sample cells, continuously or periodically
for measurement of changes in gene transcription or changes in
cellular biochemistry or structural organization. For example, one
can measure reactive oxygen species (ROIs) that are a sign of
cellular stress. One can also subject the "tissue" grown on the
porous membrane to microscopic analysis, immunohistochemical
analysis, in situ hybridization analysis, or typical pathological
analysis using staining, such as hematoxylin and eosin staining.
Samples for these analysis can be carried out in real-time, or
taken after an experiment or by taking small biopsies at different
stages during a study or an experiment.
[0042] One can subject the cells grown on the membrane to other
cells, such as immune system cells or bacterial cells, to
antibodies or antibody-directed cells, for example to target
specific cellular receptors. One can expose the cells to viruses or
other particles. To assist in detection of movement of externally
supplied substances, such as cells, viruses, particles or proteins,
one can naturally label them using typical means such as
radioactive or fluorescent labels.
[0043] Cells can be grown, cultured and analyzed using the present
device for 1, 2, 3, 4, 5, 6, or 7 days, between at least 1-2 weeks,
and even over 2 weeks. For example, as discussed below, it has been
shown that co-culture of alveolar epithelial cells with pulmonary
microvascular endothelial cells on a thin porous membrane in an
embodiment of the described device could be grown for over two
weeks without loss of viability of the cells.
[0044] The organ mimic device described herein has many different
applications including, but not limited to, identification of
markers of disease; assessing efficacy of anti-cancer therapeutics;
testing gene therapy vectors; drug development; screening; studies
of cells, especially stem cells and bone marrow cells; studies on
biotransformation, absorption, clearance, metabolism, and
activation of xenobiotics; studies on bioavailability and transport
of chemical or biological agents across epithelial or endothelial
layers; studies on transport of biological or chemical agents
across the blood-brain barrier; studies on transport of biological
or chemical agents across the intestinal epithelial barrier;
studies on acute basal toxicity of chemical agents; studies on
acute local or acute organ-specific toxicity of chemical agents;
studies on chronic basal toxicity of chemical agents; studies on
chronic local or chronic organ-specific toxicity of chemical
agents; studies on teratogenicity of chemical agents; studies on
genotoxicity, carcinogenicity, and mutagenicity of chemical agents;
detection of infectious biological agents and biological weapons;
detection of harmful chemical agents and chemical weapons; studies
on infectious diseases; studies on the efficacy of chemical or
biological agents to treat disease; studies on the optimal dose
range of agents to treat disease; prediction of the response of
organs in vivo to biological agents; prediction of the
pharmacokinetics of chemical or biological agents; prediction of
the pharmacodynamics of chemical or biological agents; studies
concerning the impact of genetic content on response to agents;
studies on gene transcription in response to chemical or biological
agents; studies on protein expression in response to chemical or
biological agents; studies on changes in metabolism in response to
chemical or biological agents. The organ mimic device can also be
used to screen on the cells, for an effect of the cells on the
materials (for example, in a manner equivalent to tissue metabolism
of a drug).
[0045] The present device may be used by one to simulate the
mechanical load environment of walking, running, breathing,
peristalsis, flow of flow or urine, or the beat of a heart, to
cells cultured from mechanically active tissues, such as heart,
lung, skeletal muscle, bone, ligament, tendon, cartilage, smooth
muscle cells, intestine, kidney, endothelial cells and cells from
other tissues. Rather than test the biological or biochemical
responses of a cell in a static environment, the investigator can
apply a range of frequencies, amplitudes and duration of mechanical
stresses, including tension, compression and shear, to cultured
cells.
[0046] A skilled artisan can implant various types of cells on the
surfaces of the membrane. Cells include any cell type from a
multicellular structure, including nematodes, amoebas, up to
mammals such as humans. Cell types implanted on the device depend
on the type of organ or organ function one wishes to mimic, and the
tissues that comprise those organs. More details of the various
types of cells implantable on the membrane of the present device
are discussed below.
[0047] One can also co-culture various stem cells, such as bone
marrow cells, induced adult stem cells, embryonal stem cells or
stem cells isolated from adult tissues on either or both sides of
the porous membrane. Using different culture media in the chambers
feeding each layer of cells, one can allow different
differentiation cues to reach the stem cell layers thereby
differentiating the cells to different cell types. One can also mix
cell types on the same side of the membrane to create co-cultures
of different cells without membrane separation.
[0048] Using the organ mimic device described herein, one can study
biotransformation, absorption, clearance, metabolism, and
activation of xenobiotics, as well as drug delivery. The
bioavailability and transport of chemical and biological agents
across epithelial layers as in the intestine, endothelial layers as
in blood vessels, and across the blood-brain barrier can also be
studied. The acute basal toxicity, acute local toxicity or acute
organ-specific toxicity, teratogenicity, genotoxicity,
carcinogenicity, and mutagenicity, of chemical agents can also be
studied. Effects of infectious biological agents, biological
weapons, harmful chemical agents and chemical weapons can also be
detected and studied. Infectious diseases and the efficacy of
chemical and biological agents to treat these diseases, as well as
optimal dosage ranges for these agents, can be studied. The
response of organs in vivo to chemical and biological agents, and
the pharmacokinetics and pharmacodynamics of these agents can be
detected and studied using the present device. The impact of
genetic content on response to the agents can be studied. The
amount of protein and gene expression in response to chemical or
biological agents can be determined. Changes in metabolism in
response to chemical or biological agents can be studied as well
using the present device.
[0049] The advantages of the organ mimic device, as opposed to
conventional cell cultures or tissue cultures, are numerous. For
instance, when cells are placed in the organ mimic device,
fibroblast, SMC (smooth muscle cell) and EC (endothelial cell)
differentiation can occur that reestablishes a defined
three-dimensional architectural tissue-tissue relationships that
are close to the in vivo situation, and cell functions and
responses to pharmacological agents or active substances or
products can be investigated at the tissue and organ levels.
[0050] In addition, many cellular or tissue activities are amenable
to detection in the organ mimic device, including, but not limited
to, diffusion rate of the drugs into and through the layered
tissues in transported flow channel; cell morphology,
differentiation and secretion changes at different layers; cell
locomotion, growth, apoptosis, and the like. Further, the effect of
various drugs on different types of cells located at different
layers of the system may be assessed easily.
[0051] For drug discovery, for example, there can be two advantages
for using the organ mimic device described herein: (1) the organ
mimic device is better able to mimic in vivo layered architecture
of tissues and therefore allow one to study drug effect at the
organ level in addition to at the cellular and tissue levels; and
(2) the organ mimic device decreases the use of in vivo tissue
models and the use of animals for drug selection and toxicology
studies.
[0052] In addition to drug discovery and development, the organ
mimic device described herein may be also useful in basic and
clinical research. For example, the organ mimic device can be used
to research the mechanism of tumorigenesis. It is well established
that in vivo cancer progression is modulated by the host and the
tumor micro-environment, including the stromal cells and
extracellular matrix (ECM). For example, stromal cells were found
being able to convert benign epithelial cells to malignant cells,
thereby ECM was found to affect the tumor formation. There is
growing evidence that cells growing in defined architecture are
more resistant to cytotoxic agents than cells in mono layers.
Therefore, a organ mimic device is a better means for simulating
the original growth characteristics of cancer cells and thereby
better reflects the real drug's sensitivity of in vivo tumors.
[0053] The organ mimic device can be employed in engineering a
variety of tissues including, but not limited to, the
cardiovascular system, lung, intestine, kidney, brain, bone marrow,
bones, teeth, and skin. If the device is fabricated with a suitable
biocompatible and/or biodegradable material, such as
poly-lactide-co-glycolide acid (PLGA), the organ mimic device may
be used for transplantation or implantation in vivo. Moreover, the
ability to spatially localize and control interactions of several
cell types presents an opportunity to engineer hierarchically, and
to create more physiologically correct tissue and organ analogs.
The arrangement of multiple cell types in defined arrangement has
beneficial effects on cell differentiation, maintenance, and
functional longevity.
[0054] The organ mimic device can also allow different growth
factors, chemicals, gases and nutrients to be added to different
cell types according to the needs of cells and their existence in
vivo. Controlling the location of those factors or proteins may
direct the process of specific cell remodeling and functioning, and
also may provide the molecular cues to the whole system, resulting
in such beneficial developments as neotissue, cell remodeling,
enhanced secretion, and the like.
[0055] In yet another aspect, the organ mimic device can be
utilized as multi cell type cellular microarrays, such as
microfluidic devices. Using the organ mimic device, pattern
integrity of cellular arrays can be maintained. These cellular
microarrays may constitute the future "lab-on-a-chip", particularly
when multiplexed and automated. These miniaturized multi cell type
cultures will facilitate the observation of cell dynamics with
faster, less noisy assays, having built-in complexity that will
allow cells to exhibit in vivo-like responses to the array.
[0056] In yet another aspect, the organ mimic device can be
utilized as biological sensors. Cell-based biosensors can provide
more information than other biosensors because cells often have
multifaceted physiological responses to stimuli, as well as novel
mechanisms to amplify these responses. All cell types in the organ
mimic device can be used to monitor different aspects of an analyte
at the same time; different cell type in the organ mimic device can
be used to monitor different analytes at the same time; or a
mixture of both types of monitoring. Cells ranging from E. coli to
cells of mammalian lines have been used as sensors for applications
in environmental monitoring, toxin detection, and physiological
monitoring.
[0057] In yet another aspect, the organ mimic device can be used in
understanding fundamental processes in cell biology and cell-ECM
interactions. The in vivo remodeling process is a complicated,
dynamic, reciprocal process between cells and ECMs. The organ mimic
device would be able to capture the complexity of these biological
systems, rendering these systems amenable to investigation and
beneficial manipulation. Furthermore, coupled with imaging tools,
such as fluorescence microscopy, microfluorimetry or optical
coherence tomography (OCT), real-time analysis of cellular behavior
in the multilayered tissues is expected using the device. Examples
of cell and tissue studies amenable to real-time analysis include
cell secretion and signaling, cell-cell interactions, tissue-tissue
interactions, dynamic engineered tissue construction and
monitoring, structure-function investigations in tissue
engineering, and the process of cell remodeling matrices in
vitro.
[0058] Another example of the use of this device is to induce
tissue-tissue interfaces and complex organ structures to form
within the device by implanting it in vivo within the body of a
living animal, and allowing cells and tissues to impregnate the
device and establish normal tissue-tissue interfaces. Then the
whole device and contained cells and tissues is surgically removed
while perfusing it through one or more of the fluid channels with
medium and gases necessary for cell survival. This complex organ
mimic may then be maintained viable in vitro through continuous
perfusion and used to study highly complex cell and tissue
functions in their normal 3D context with a level of complexity not
possible using any existing in vitro model system.
[0059] In particular, a microchannel device may be implanted
subcutaneously in vivo into an animal in which the device contains
bone-inducing materials, such as demineralized bone powder or bone
morphogenic proteins (BMPs), in a channel that has one or more
corresponding ports open to the surrounding tissue space. The
second channel would be closed during implantation by closing its
end ports or filling it with a solid removable materials, such as a
solid rod. As a result of wound healing, connective tissues
containing microcapillaries and mesenchymal stem cells would grow
into the open channels of the device and, due to the presence of
the bone-inducing material, will form bone with spaces that recruit
circulating hematopoietic precursor cells to form fully functional
bone marrow, as shown in past studies.
[0060] Once this process is complete, the surgical site would be
reopened, and the second channel would be reopened by removing the
rod or plugs and would then be connected with catheters linked to a
fluid reservoir so that culture medium containing nutrients and
gases necessary for cell survival could be pumped through the
second channel and passed through the pores of the membrane into
the first channel containing the formed bone marrow. The entire
microchannel device could then be cut free from the surrounding
tissue, and with the medium flowing continuously into the device,
would be removed from the animal and passed to a tissue culture
incubator and maintained in culture with continuous fluid flow
through the second channel, and additional flow can be added to the
first channel as well if desired by connecting to their inlet and
outlet ports. The microchannel device would then be used to study
intact bone marrow function in vitro as in a controlled
environment.
[0061] Both biocompatible and biodegradable materials can be used
in the present device to facilitate in vivo implantation of the
present device. One can also use biocompatible and biodegradable
coatings. For example, one can use ceramic coatings on a metallic
substrate. But any type of coating material and the coating can be
made of different types of materials: metals, ceramics, polymers,
hydrogels or a combination of any of these materials.
[0062] Biocompatible materials include, but are not limited to an
oxide, a phosphate, a carbonate, a nitride or a carbonitride. Among
the oxide the following ones are preferred: tantalum oxide,
aluminum oxide, iridium oxide, zirconium oxide or titanium oxide.
In some cases the coating can also be made of a biodegradable
material that will dissolve over time and may be replaced by the
living tissue. Substrates are made of materials such as metals,
ceramics, polymers or a combination of any of these. Metals such as
stainless steel, Nitinol, titanium, titanium alloys, or aluminum
and ceramics such as zirconia, alumina, or calcium phosphate are of
particular interest.
[0063] The biocompatible material can also be biodegradable in that
it will dissolve over time and may be replaced by the living
tissue. Such biodegradable materials include, but are not limited
to, poly(lactic acid-co-glycolic acid), polylactic acid,
polyglycolic acid (PGA), collagen or other ECM molecules, other
connective tissue proteins, magnesium alloys, polycaprolactone,
hyaluric acid, adhesive proteins, biodegradable polymers,
synthetic, biocompatible and biodegradable material, such as
biopolymers, bioglasses, bioceramics, calcium sulfate, calcium
phosphate such as, for example, monocalcium phosphate monohydrate,
monocalcium phosphate anhydrous, dicalcium phosphate dihydrate,
dicalcium phosphate anhydrous, tetracalcium phosphate, calcium
orthophosphate phosphate, calcium pyrophosphate, alpha-tricalcium
phosphate, beta-tricalcium phosphate, apatite such as
hydroxyapatite, or polymers such as, for example,
poly(alpha-hydroxyesters), poly(ortho esters), poly(ether esters),
polyanhydrides, poly(phosphazenes), poly(propylene fumarates),
poly(ester amides), poly(ethylene fumarates), poly(amino acids),
polysaccharides, polypeptides, poly(hydroxy butyrates),
poly(hydroxy valerates), polyurethanes, poly(malic acid),
polylactides, polyglycolides, polycaprolactones,
poly(glycolide-co-trimethylene carbonates), polydioxanones, or
copolymers, terpolymers thereof or blends of those polymers, or a
combination of biocompatible and biodegradable materials. One can
also use biodegradable glass and bioactive glass self-reinforced
and ultrahigh strength bioabsorbable composites assembled from
partially crystalline bioabsorbable polymers, like polyglycolides,
polylactides and/or glycolide/lactide copolymers.
[0064] These materials preferably have high initial strength,
appropriate modulus and strength retention time from 4 weeks up to
1 year in vivo, depending on the implant geometry. Reinforcing
elements such as fibers of crystalline polymers, fibers of carbon
in polymeric resins, and particulate fillers, e.g., hydroxyapatite,
may also be used to provide the dimensional stability and
mechanical properties of biodegradable devices. The use of
interpenetrating networks (IPN) in biodegradable material
construction has been demonstrated as a means to improve mechanical
strength. To further improve the mechanical properties of
IPN-reinforced biodegradable materials, the present device may be
prepared as semi-interpenetrating networks (SIPN) of crosslinked
polypropylene fumarate within a host matrix of
poly(lactide-co-glycolide) 85:15 (PLGA) or
poly(1-lactide-co-d,l-lactide) 70:30 (PLA) using different
crosslinking agents. One can also use natural
poly(hydroxybutyrate-co-9% hydroxyvalerate) copolyester membranes
as described in Charles-Hilaire Rivard et al. (Journal of Applied
Biomaterials, Volume 6 Issue 1, Pages 65-68, 1 Sep. 2004). A
skilled artisan will be able to also select other biodegradable
materials suitable for any specific purposes and cell and tissue
types according to the applications in which the device is
used.
[0065] The device as described can also be used as therapeutic
devices, when placed in vivo. One can re-create organ mimics, such
as bone marrow or lymph nodes by placing the devices in, for
example an animal model allowing the device to be inhabited by
living cells and tissues, and then removing the entire device with
living cells while perfusing the vascular channel with medium. The
device can then be removed and kept alive ex vivo for in vitro or
ex vivo studies. In particular, the membrane may be coated with one
or more cell layers on at least one side of the membrane in vitro.
In this embodiment, the cells are plated outside an organism. In an
embodiment, the membrane is coated with one or more cell layers on
at least one side of the membrane in vivo. One can treat one side
of the membrane in vitro and the other side in vivo. One can also
have one or both sides initially coated with one cell type in vitro
and then implant the device to attract additional cell layers in
vivo.
[0066] In general, the present disclosure is directed to device and
method of use in which the device includes a body having a central
microchannel separated by one or more porous membranes. The
membrane(s) are configured to divide the central microchannel into
two or more closely apposed parallel central microchannels, wherein
one or more first fluids are applied through the first central
microchannel and one or more second fluids are applied through the
second or more central microchannels. The surfaces of each porous
membrane can be coated with cell adhesive molecules to support the
attachment of cells and promote their organization into tissues on
the upper and lower surface of the membrane, thereby creating a
tissue-tissue interface separated by a porous membrane between the
adjacent parallel fluid channels. The membrane may be porous,
flexible, elastic, or a combination thereof with pores large enough
to only permit exchange of gases and small chemicals, or large
enough to permit migration and transchannel passage of large
proteins, and whole living cells. Fluid pressure, flow and channel
geometry also may be varied to apply a desired fluid shear stress
to one or both tissue layers.
[0067] In a non-limiting example embodiment, the device is
configured to mimic operation of a lung, whereby lung epithelium
cells self assemble on one surface of the ECM membrane and lung
capillary endothelium cells self assemble on the opposite face of
the same porous membrane. The device thereby allows simulation of
the structure and function of a functional alveolar-capillary unit
that can be exposed to physiological mechanical strain to simulate
breathing or to both air-borne and blood-borne chemical, molecular,
particulate and cellular stimuli to investigate the exchange of
chemicals, molecules, and cells across this tissue-tissue interface
through the pores of the membrane. The device impacts the
development of in vitro lung models that mimic organ-level
responses which are able to be analyzed under physiological and
pathological conditions. This system may be used in several
applications including, but not limited to, drug screening, drug
delivery, vaccine delivery, biodetection, toxicology, physiology
and organ/tissue engineering applications.
[0068] FIG. 1 illustrates a block diagram of the overall system
employing the inventive device in accordance with an embodiment. As
shown in FIG. 1, the system 100 includes an organ mimic device 102,
one or more fluid sources 104, 104.sub.N coupled to the device 102,
one or more optional pumps 106 coupled to the fluid source 104 and
device 102. One or more CPUs 110 are coupled to the pump 106 and
preferably control the flow of fluid in and out of the device 102.
The CPU preferably includes one or processors 112 and one or more
local/remote storage memories 114. A display 116 is coupled to the
CPU 110, and one or more pressure sources 118 are coupled to the
CPU 110 and the device 102. The CPU 110 preferably controls the
flow and rate of pressurized fluid to the device. It should be
noted that although one interface device 102 is shown and described
herein, it is contemplated that a plurality of interface devices
102 may be tested and analyzed within the system 100 as discussed
below.
[0069] As will be discussed in more detail, the organ mimic device
102 preferably includes two or more ports which place the
microchannels of the device 102 in communication with the external
components of the system, such as the fluid and pressure sources.
In particular, the device 102 is coupled to the one or more fluid
sources 104.sub.N in which the fluid source may contain air, blood,
water, cells, compounds, particulates, and/or any other media which
are to be delivered to the device 102. In an embodiment, the fluid
source 104 provides fluid to one or more microchannels of the
device 102 and also preferably receives the fluid which exits the
device 102. It is contemplated that the fluid exiting the device
102 may additionally or alternatively be collected in a fluid
collector or reservoir 108 separate from the fluid source 104.
Thus, it is possible that separate fluid sources 104, 104.sub.N
respectively provide fluid to and remove fluid from the device
102.
[0070] In an embodiment, fluid exiting the device 102 may be reused
and reintroduced into the same or different input port through
which it previously entered. For example, the device 102 may be set
up such that fluid passed through a particular central microchannel
is recirculated back to the device and is again run through the
same central microchannel. This could be used, for instance, to
increase the concentration of an analyte in the fluid as it is
recirculated the device. In another example, the device 102 may be
set up such that fluid passed through the device and is
recirculated back into the device and then subsequently run through
another central microchannel. This could be used to change the
concentration or makeup of the fluid as it is circulated through
another microchannel.
[0071] One or more pumps 106 are preferably utilized to pump the
fluid into the device 102, although pumps in general are optional
to the system. Fluid pumps are well known in the art and are not
discussed in detail herein. As will be discussed in more detail
below, each microchannel portion is preferably in communication
with its respective inlet and/or outlet port, whereby each
microchannel portion of allow fluid to flow therethrough.
[0072] Each microchannel in the device preferably has dedicated
inlet and outlet ports which are connected to respective dedicated
fluid sources and/or fluid collectors to allow the flow rates, flow
contents, pressures, temperatures and other characteristics of the
media to be independently controlled through each central
microchannel. Thus, one can also monitor the effects of various
stimuli to each of the cell or tissue layers separately by sampling
the separate fluid channels for the desired cellular marker, such
as changes in gene expression at RNA or protein level.
[0073] The cell injector/remover 108 component is shown in
communication with the device 102, whereby the injector/remover 108
is configured to inject, remove and/or manipulate cells, such as
but not limited to epithelial and endothelial cells, on one or more
surfaces of the interface membrane within the device 102
independent of cells introduced into the device via the inlet
port(s) 210, 218. For example, blood containing magnetic particles
which pull pathogenic cells may be cultured in a separate device
whereby the mixture can be later introduced into the system via the
injector at a desired time without having to run the mixture
through the fluid source 104. In an embodiment, the cell
injector/remover 108 is independently controlled, although the
injector/remover 108 may be controlled by the CPU 110 as shown in
FIG. 1. The cell injector/remover 108 is an optional component and
is not necessary.
[0074] Although not required, pressure may be applied from the one
or more pressure sources 118 to create a pressure differential to
cause mechanical movements within the device 102. In an embodiment
in which pressures are used with the device, the pressure source
118 is controlled by the CPU 110 to apply a pressure differential
within the device to effectively cause one or more membranes (FIGS.
3A-3B) within the device to expand and/or contract in response to
the applied pressure differential. In an embodiment, the pressure
applied to the device 100 by the pressure source 118 is a positive
pressure, depending on the configuration or application of the
device. Additionally or alternatively, the pressure applied by the
pressure source 118 is a negative pressure, such as vacuum or
suction, depending on the configuration or application of the
device. The pressure source 118 is preferably controlled by the CPU
110 to apply pressure at set timed intervals or frequencies to the
device 102, whereby the timing intervals may be set to be uniform
or non-uniform. The pressure source 118 may be controlled to apply
uniform pressure in the timing intervals or may apply different
pressures at different intervals. For instance, the pressure
applied by the pressure source 118 may have a large magnitude
and/or be set at a desired frequency to mimic a person running or
undergoing exertion. The pressure source 118 may also apply slow,
irregular patterns, such as simulating a person sleeping. In an
embodiment, the CPU 110 operates the pressure source 118 to
randomly vary intervals of applying pressure to cause cyclic
stretching patterns to simulate irregularity in breath rate and
tidal volumes during natural breathing.
[0075] One or more sensors 120 may be coupled to the device 102 to
monitor one or more areas within the device 102, whereby the
sensors 120 provide monitoring data to the CPU 110. One type of
sensor 120 is preferably a pressure sensor which provides data
regarding the amount of pressure in one or more operating or
central microchannels the device 102. Pressure data from opposing
sides of the microchannel walls may be used to calculate real-time
pressure differential information between the operating and central
microchannels. The monitoring data would be used by the CPU 110 to
provide information on the device's operational conditions as well
as how the cells are behaving within the device 102 in particular
environments in real time. The sensor 120 may be an electrode, have
infrared, optical (e.g. camera, LED), or magnetic capabilities or
utilize any other appropriate type of technology to provide the
monitoring data. For instance, the sensor may be one or more
microelectrodes which analyze electrical characteristics across the
membrane (e.g. potential difference, resistance, and short circuit
current) to confirm the formation of an organized barrier, as well
as its fluid/ion transport function across the membrane. It should
be noted that the sensor 120 may be external to the device 102 or
be integrated within the device 102. It is contemplated that the
CPU 110 controls operation of the sensor 120, although it is not
necessary. The data is preferably shown on the display 116.
[0076] FIG. 2A illustrates a perspective view of the tissue
interface device in accordance with an embodiment. In particular,
as shown in FIG. 2A, the device 200 (also referred to reference
numeral 102) preferably includes a body 202 having a branched
microchannel design 203 in accordance with an embodiment. The body
202 may be made of a flexible material, although it is contemplated
that the body be alternatively made of a non-flexible material. It
should be noted that the microchannel design 203 is only exemplary
and not limited to the configuration shown in FIG. 2A. The body 202
is preferably made of a flexible biocompatible polymer, including
but not limited to, polydimethyl siloxane (PDMS), or polyimide. It
is also contemplated that the body 202 may be made of non-flexible
materials like glass, silicon, hard plastic, and the like. Although
it is preferred that the interface membrane be made of the same
material as the body 202, it is contemplated that the interface
membrane be made of a material that is different than the body of
the device.
[0077] The device in FIG. 2A includes a plurality of ports 205
which will be described in more detail below. In addition, the
branched configuration 203 includes a tissue-tissue interface
simulation region (membrane 208 in FIG. 2B) where cell behavior
and/or passage of gases, chemicals, molecules, particulates and
cells are monitored. FIG. 2B illustrates an exploded view of the
organ mimic device in accordance with an embodiment. In particular,
the outer body 202 of the device 200 is preferably comprised of a
first outer body portion 204, a second outer body portion 206 and
an intermediary porous membrane 208 configured to be mounted
between the first and second outer body portions 204, 206 when the
portions 204, 206 are mounted to one another to form the overall
body.
[0078] FIG. 2B illustrates an exploded view of the device in
accordance with an embodiment. As shown in FIG. 2B, the first outer
body portion 204 includes one or more inlet fluid ports 210
preferably in communication with one or more corresponding inlet
apertures 211 located on an outer surface of the body 202. The
device 100 is preferably connected to the fluid source 104 via the
inlet aperture 211 in which fluid travels from the fluid source 104
into the device 100 through the inlet fluid port 210.
[0079] Additionally, the first outer body portion 204 includes one
or more outlet fluid ports 212 preferably in communication with one
or more corresponding outlet apertures 215 on the outer surface of
the body 202. In particular, fluid passing through the device 100
exits the device 100 to a fluid collector 108 or other appropriate
component via the corresponding outlet aperture 215. It should be
noted that the device 200 may be set up such that the fluid port
210 is an outlet and fluid port 212 is an inlet. Although the inlet
and outlet apertures 211, 215 are shown on the top surface of the
body 202, one or more of the apertures may be located on one or
more sides of the body.
[0080] In an embodiment, the inlet fluid port 210 and the outlet
fluid port 212 are in communication with the first central
microchannel 250A (see FIG. 3A) such that fluid can dynamically
travel from the inlet fluid port 210 to the outlet fluid port 212
via the first central microchannel 250A, independently of the
second central microchannel 250B (see FIG. 3A).
[0081] It is also contemplated that the fluid passing between the
inlet and outlet fluid ports may be shared between the central
sections 250A and 250B. In either embodiment, characteristics of
the fluid flow, such as flow rate and the like, passing through the
central microchannel 250A is controllable independently of fluid
flow characteristics through the central microchannel 250B and vice
versa.
[0082] In addition, the first portion 204 includes one or more
pressure inlet ports 214 and one or more pressure outlet ports 216
in which the inlet ports 214 are in communication with
corresponding apertures 217 located on the outer surface of the
device 100. Although the inlet and outlet apertures are shown on
the top surface of the body 202, one or more of the apertures may
alternatively be located on one or more sides of the body.
[0083] In operation, one or more pressure tubes (not shown)
connected to the pressure source 118 (FIG. 1) provides positive or
negative pressure to the device via the apertures 217.
Additionally, pressure tubes (not shown) are connected to the
device 100 to remove the pressurized fluid from the outlet port 216
via the apertures 223. It should be noted that the device 200 may
be set up such that the pressure port 214 is an outlet and pressure
port 216 is an inlet. It should be noted that although the pressure
apertures 217, 223 are shown on the top surface of the body 202, it
is contemplated that one or more of the pressure apertures 217, 223
may be located on one or more side surfaces of the body 202.
[0084] Referring to FIG. 2B, the second outer body portion 206
preferably includes one or more inlet fluid ports 218 and one or
more outlet fluid ports 220. It is preferred that the inlet fluid
port 218 is in communication with aperture 219 and outlet fluid
port 220 is in communication with aperture 221, whereby the
apertures 219 and 221 are preferably located on the outer surface
of the second outer body portion 206. Although the inlet and outlet
apertures are shown on the surface of the body 202, one or more of
the apertures may be alternatively located on one or more sides of
the body.
[0085] As with the first outer body portion 204 described above,
one or more fluid tubes connected to the fluid source 104 (FIG. 1)
are preferably coupled to the aperture 219 to provide fluid to the
device 100 via port 218. Additionally, fluid exits the device 100
via the outlet port 220 and out aperture 221 to a fluid
reservoir/collector 108 or other component. It should be noted that
the device 200 may be set up such that the fluid port 218 is an
outlet and fluid port 220 is an inlet.
[0086] In addition, it is preferred that the second outer body
portion 206 includes one or more pressure inlet ports 222 and one
or more pressure outlet ports 224. In particular, it is preferred
that the pressure inlet ports 222 are in communication with
apertures 227 and pressure outlet ports 224 are in communication
with apertures 229, whereby apertures 227 and 229 are preferably
located on the outer surface of the second portion 206. Although
the inlet and outlet apertures are shown on the bottom surface of
the body 202, one or more of the apertures may be alternatively
located on one or more sides of the body. Pressure tubes connected
to the pressure source 118 (FIG. 1) are preferably engaged with
ports 222 and 224 via corresponding apertures 227 and 229. It
should be noted that the device 200 may be set up such that the
pressure port 222 is an outlet and fluid port 224 is an inlet.
[0087] In an embodiment, the membrane 208 is mounted between the
first portion 204 and the second portion 206, whereby the membrane
208 is located within the body 202 of the device 200 (see FIG. 5E).
In an embodiment, the membrane 208 is a made of a material having a
plurality of pores or apertures therethrough, whereby molecules,
cells, fluid or any media is capable of passing through the
membrane 208 via one or more pores in the membrane 208. As
discussed in more detail below, it is contemplated in an embodiment
that the porous membrane 208 may be made of a material which allows
the membrane 208 to undergo stress and/or strain in response to
pressure differentials present between the central microchannels
250A, 250B and the operating microchannels. Alternatively, the
porous membrane 208 is relatively inelastic in which the membrane
208 undergoes minimal or no movement while media is passed through
one or more of the central microchannels 250A, 250B and cells
organize and move between the central microchannels 250A, 250B via
the porous membrane.
[0088] Referring FIG. 2C illustrates a perspective view of the
tissue-tissue interface region of the first outer portion 204 of
the body taken at line C-C (from FIG. 2B). As shown in FIG. 2C, the
top portion of the tissue-tissue interface region 207A is within
the body of the first portion 204 and includes a top portion of a
central microchannel 230 and one or more top portion side operating
microchannels 232 located adjacent to the central microchannel 230.
Microchannel walls 234 preferably separate the central microchannel
230 from the operating microchannels 232 such that fluid traveling
through the central microchannel 230 does not pass into operating
microchannels 232. Likewise, the channel walls 234 prevent
pressurized fluid passing along operating microchannels 232 from
entering the central microchannel 230. It should be noted that a
pair of operating microchannels 232 are shown on opposing sides of
central microchannel 230 in FIGS. 2C and 3A, however it is
contemplated that the device may incorporate more than two
operating microchannels 232. It is also contemplated that the
device 200 may include only one operating microchannel 232 adjacent
to the central microchannel 230.
[0089] FIG. 2D illustrates a perspective view of the tissue
interface region taken at line D-D of the second outer portion 206
of the body. As shown in FIG. 2D, the tissue interface region
includes a bottom portion of the central microchannel 240 and at
least two bottom portions of operating microchannels 242 located
adjacent to the central microchannel 240 portion. A pair of channel
walls 234 preferably separate the central microchannel 240 from the
operating microchannels 232 such that fluid traveling through the
central microchannel 230 does not pass into operating microchannels
232. Likewise, the channel walls 234 prevent pressurized fluid
passing along operating microchannels 232 from entering the central
microchannel 230.
[0090] As shown in FIGS. 2C and 2D, the top and bottom portions 230
and 240 of the central microchannel each have a range of width
dimension (shown as B) between 50 and 1000 microns, and preferably
around 400 microns. It should be noted that other width dimensions
are contemplated depending on the type of physiological system
which is being mimicked in the device. Additionally, the top and
bottom portions of the operating microchannels 232 and 242 each
have a width dimension (shown as A) between 25 and 800 microns, and
preferably around 200 microns, although other width dimensions are
contemplated. The height dimensions of the central and/or operating
microchannels range between 50 microns and several centimeters, and
preferably around 200 microns. The microchannel walls 234, 244
preferably have a thickness range between 5 microns to 50 microns,
although other width dimensions are contemplated depending on the
material used for the walls, application in which the device is
used and the like.
[0091] FIG. 3A illustrates a perspective view of the tissue
interface region within the body in accordance with an embodiment.
In particular, FIG. 3A illustrates the first portion 207A and the
second portion 207B mated to one another whereby the side walls 228
and 238 as well as channel walls 234, 244 form the overall central
microchannel 250 and operating microchannels 252. As stated above,
it is preferred that central microchannel 250 and operating
microchannels 252 are separated by the walls 234, 244 such that
fluid is not able to pass between the channels 250, 252.
[0092] The membrane 208 is preferably positioned in the center of
the central microchannel 250 and is oriented along a plane parallel
to the x-y plane shown in FIG. 3A. It should be noted that although
one membrane 208 is shown in the central microchannel 250, more
than one membrane 208 may be configured within the central
microchannel 250, as discussed in more detail below. In addition to
being positioned within the central microchannel 250, the membrane
208 is sandwiched in place by channel walls 234, 244 during
formation of the device.
[0093] The membrane 208 preferably separates the overall central
microchannel 250 into two or more distinct central microchannels
250A and 250B. It should be noted that although the membrane 208 is
shown midway through the central microchannel 250, the membrane 208
may alternatively be positioned vertically off-center within the
central microchannel 250, thus making one of the central
microchannel sections 250A, 250B larger in volume or cross-section
than the other microchannel section.
[0094] As will be discussed in more detail below, the membrane 208
may have at least a portion which is porous to allow cells or
molecules to pass therethrough. Additionally or alternatively, at
least a portion of the membrane 208 may have elastic or ductile
properties which allow the membrane 208 to be manipulated to
expand/contract along one or more planar axe. Thus, it is
contemplated that one or more portions of the membrane 208 may be
porous and elastic or porous, but inelastic.
[0095] With regard to the porous and elastic membrane, a pressure
differential may be applied within the device to cause relative
continuous expansion and contraction of the membrane 208 along the
x-y plane. In particular, as stated above, one or more pressure
sources preferably apply pressurized fluid (e.g. air) along the one
or more operating microchannels 252, whereby the pressurized fluid
in the microchannels 252 creates a pressure differential on the
microchannel walls 234, 244. The membrane 208 may have an
elasticity depending on the type of material that it is made of. If
the membrane 208 is made of more than one material, the weight
ratio of the respective materials which make up the membrane is a
factor in determining the elasticity. For example, in the
embodiment that the membrane 208 is made of PDMS, the Young's
modulus values are in the ranges of 12 kPa-20 MPa, although other
elasticity values are contemplated.
[0096] In the embodiments shown in FIGS. 3A and 3B, the pressurized
fluid is a vacuum or suction force that is applied only through the
operating microchannels 252. The difference in pressure caused by
the suction force against the microchannel walls 234, 244 causes
the walls 234, 244 to bend or bulge outward toward the sides of the
device 228, 238 (see FIG. 3B). Considering that the membrane 208 is
mounted to and sandwiched between the walls 234, 244, the relative
movement of the walls 234, 244 thereby causes the opposing ends of
the membrane to move along with the walls to stretch (shown as 208'
in FIG. 3B) along the membrane's plane. This stretching mimics the
mechanical forces experienced by a tissue-tissue interface, for
example, in the lung alveolus during breathing, and thus provides
the important regulation for cellular self assembly into tissue
structures and cell behavior.
[0097] When the negative pressure is no longer applied (and/or
positive pressure is applied to the operating channels), the
pressure differential between the operating channels 252 and the
central channel 250 decreases and the channel walls 234, 244
retract elastically toward their neutral position (as in FIG. 3A).
During operation, the negative pressure is alternately applied in
timed intervals to the device 200 to cause continuous expansion and
contraction of the membrane 208 along its plane, thereby mimicking
operation of the tissue-tissue interface of the living organ within
a controlled in vitro environment. As will be discussed, this
mimicked organ operation within the controlled environment allows
monitoring of cell behavior in the tissues, as well as passage of
molecules, chemicals, particulates and cells with respect to the
membrane and the associated first and second microchannels 250A,
250B.
[0098] It should be noted that the term pressure differential in
the present specification relates to a difference of pressure on
opposing sides of a particular wall between the central
microchannel and the outer operating channel. It is contemplated
that the pressure differential may be created in a number of ways
to achieve the goal of expansion and/or contraction of the membrane
208. As stated above, a negative pressure (i.e. suction or vacuum)
may be applied to one or more of the operating channels 252.
Alternatively, it is contemplated that the membrane 208 is
pre-loaded or pre-stressed to be in an expanded state by default
such that the walls 234, 244 are already in the bent configuration,
as show in FIG. 3B. In this embodiment, positive pressure applied
to the operating channel 252 will create the pressure differential
which causes the walls 234, 244 to move inward toward the central
microchannel (see in FIG. 3A) to contract the membrane 208.
[0099] It is also contemplated, in another embodiment, that a
combination of positive and negative pressure is applied to one or
more operating microchannels 252 to cause movement of the membrane
208 along its plane in the central microchannel. In any of the
above embodiments, it is desired that the pressure of the fluid in
the one or more operating channels 252 be such that a pressure
differential is in fact created with respect to the pressure of the
fluid(s) in one or more of the central microchannel(s) 250A, 250B
to cause relative expansion/contraction of the membrane 208. For
example, fluid may have a certain pressure may be applied within
the top central microchannel 250A, whereby fluid in the bottom
central microchannel 250B may have a different pressure. In this
example, pressure applied to the one or more operating channels 252
must take into account the pressure of the fluid in either or both
of the central microchannels 250A, 250B to ensure desired
expansion/contraction of the membrane 208.
[0100] It is possible, in an embodiment, for a pressure
differential to exist between the top and bottom microchannels
250A, 250B to cause at least a portion of the membrane 208 to
expand and/or contract vertically in the z-direction in addition to
expansion/contraction along the x-y plane.
[0101] In an embodiment, the expansion and retraction of the
membrane 208 preferably applies mechanical forces to the adherent
cells and ECM that mimic physiological mechanical cues that can
influence transport of chemicals, molecules particulates, and/or
fluids or gas across the tissue-tissue interface, and alter cell
physiology. It should be noted that although pressure differentials
created in the device preferably cause expansion/contraction of the
membrane, it is contemplated that mechanical means, such as
micromotors or actuators, may be employed to assist or substitute
for the pressure differential to cause expansion/contraction of the
cells on the membrane to modulatecell physiology.
[0102] FIGS. 3E and 4C illustrate perspectives view of the membrane
208 which includes a plurality of apertures 302 extending
therethrough in accordance with an embodiment. In particular, the
membrane shown in FIGS. 3E and 4C includes one or more of
integrated pores or apertures 302 which extend between a top
surface 304 and a bottom surface 306 of the membrane 208.
[0103] The membrane is configured to allow cells, particulates,
chemicals and/or media to migrate between the central microchannel
portions 250A, 250B via the membrane 208 from one section of the
central microchannel to the other or vice versa. The pore apertures
are shown to have a pentagonal cross sectional shape in FIGS.
4A-4C, although any other cross sectional shape is contemplated,
including but not limited to, a circular shaped 302, hexagonal 308,
square, elliptical 310 and the like. The pores 302, 308, 310
(generally referred to as reference numeral 302) preferably extend
vertically between the top and bottom surfaces 304, 306, although
it is contemplated that they may extend laterally as well between
the top and bottom surfaces, as with pore 312. It should also be
noted that the porous may additionally/alternatively incorporate
slits or other shaped apertures along at least a portion of the
membrane 208 which allow cells, particulates, chemicals and/or
fluids to pass through the membrane 208 from one section of the
central microchannel to the other.
[0104] The width dimension of the pores are preferably in the range
of 0.5 microns and 20 microns, although it is preferred that the
width dimension be approximately 10 microns. It is contemplated,
however, that the width dimension be outside of the range provided
above. In some embodiments, the membrane 208 has pores or apertures
larger than traditional molecular/chemical filtration devices,
which allow cells as well as molecules to migrate across the
membrane 208 from one microchannel section (e.g. 250A) to the other
microchannel section (e.g. 250B) or vice versa. This may be useful
in culturing cells which polarize in the top and bottom central
channels in response to being in the microchannel environment
provided by the device whereby fluid(s) and cells are dynamically
passed through pores that connect these microchannels 250A,
250B.
[0105] As shown in FIG. 4B, the thickness of the membrane 208 may
be between 70 nanometers and 50 microns, although a preferred range
of thickness would between 5 and 15 microns. It is also
contemplated that the membrane 208 be designed to include regions
which have lesser or greater thicknesses than other regions in the
membrane 208. As shown in FIG. 3C, the membrane 208 is shown to
have one or more decreased thickness areas 209 relative to the
other areas of the membrane 208. The decreased thickness area(s)
209 may run along the entire length or width of the membrane 208 or
may alternatively be located at only certain locations of the
membrane 208. It should be noted that although the decreased
thickness area 209 is shown along the bottom surface of the
membrane 208 (i.e. facing microchannel 250B), it is contemplated
that the decreased thickness area(s) 209 may
additionally/alternatively be on the opposing surface of the
membrane 208 (i.e. facing microchannel 250A). It should also be
noted that at least portions of the membrane 208 may have one or
more larger thickness areas 209' relative to the rest of the
membrane, as shown in FIG. 3D and capable of having the same
alternatives as the decreased thickness areas described above.
[0106] In an embodiment, the porous membrane 208 may be designed or
surface patterned to include micro and/or nanoscopic patterns
therein such as grooves and ridges, whereby any parameter or
characteristic of the patterns may be designed to desired sizes,
shapes, thicknesses, filling materials, and the like.
[0107] In an embodiment, the membrane 208 is made of
polydimethylsiloxane (PDMS) or any other polymeric compound or
material, although this is not necessary. For instance, the
membrane 208 may be made of polyimide, polyester, polycarbonate,
cyclicolefin copolymer, polymethylmethacrylate, nylon,
polyisoprene, polybutadiene, polychlorophene, polyisobutylene,
poly(styrene-butadiene-styrene), nitriles, the polyurethanes and
the polysilicones. GE RTV 615, a vinyl-silane crosslinked (type)
silicone elastomer (family) may be used. Polydimethysiloxane (PDMS)
membranes are available HT-6135 and HT-6240 membranes from Bisco
Silicons (Elk Grove, Ill.) and are useful in selected applications.
The choice of materials typically depends upon the particular
material properties (e.g., solvent resistance, stiffness, gas
permeability, and/or temperature stability) required for the
application being conducted. Additional elastomeric materials that
can be used in the manufacture of the components of the
microfluidic devices described in Unger et al., (2000 Science
288:113-116). Some elastomers of the present devices are used as
diaphragms and in addition to their stretch and relax properties,
are also selected for their porosity, impermeability, chemical
resistance, and their wetting and passivating characteristics.
Other elastomers are selected for their thermal conductivity.
Micronics Parker Chomerics Thermagap material 61-02-0404-F574
(0.020'' thick) is a soft elastomer (<5 Shore A) needing only a
pressure of 5 to 10 psi to provide a thermal conductivity of 1.6
W/m-.degree. K. Deformable films, lacking elasticity, can also be
used in the microfluidic device. One may also use silk, ECM gels
with or without crosslinking as other such suitable materials to
make the devices and membranes as described.
[0108] It should be noted that although the central and operating
microchannels 250, 252 are shown to have substantially square or
rectangular cross sections, other cross-sectional shapes are
contemplated such as circular, oval, hexagonal, and the like. It is
also contemplated that the device 200 may have more or less than
two operating channels 252 and/or more or less than two central
microchannels 250A, 250B in accordance with an embodiment.
[0109] In an embodiment, it is contemplated that the central
microchannel has a non-uniform width dimension B along at least a
portion of its length in the device. FIG. 2E illustrates a cross
sectional top-down view of the tissue interface region 400 in
accordance with an embodiment. As shown in FIG. 2E, the interface
400 includes the central microchannel 402 along with adjacent
operating channels 406 separated by microchannel walls 404. In the
embodiment in FIG. 2E, the central microchannel 402 is shown to
have a gradually increasing width from width dimension C (at end
408) to width dimension D (at end 410). In the embodiment in FIG.
2E, the operating channels 406 each have a correspondingly
decreasing width dimension (from width dimension E at end 408 to
width dimension F at end 410). In another embodiment, as shown in
FIG. 2F, the operating channels 406' have a substantially uniform
width dimension F at ends 408 and 410. It is contemplated that the
membrane (not shown) be placed above the central microchannel 402
and mounted to the top surface of the walls 404, whereby the
membrane has a tapered shape similar to the central microchannel
402. The tapered membrane would thereby undergo non-uniform
stretching in the direction of the arrows when the pressure
differential is applied between the operating microchannels 406 and
the central microchannel 402.
[0110] In another embodiment, the central microchannel may have a
portion which has a partially circular cross sectional shape, as
shown in FIG. 2G. In particular to the embodiment in FIG. 2G, a
pressure differential created between the central microchannel 502
and the adjacent operating microchannels 504 will cause the
microchannel walls 506 to move in the direction represented by the
arrows. With regard to the circular portion 508 of the central
microchannel 502, equiaxial outward movement of the walls (as shown
by the arrows) at the central portion 508 produces equiaxial
stretching of the membrane (not shown) mounted atop of the walls
506.
[0111] The device 200 described herein has potential for several
applications. For example, in one application, the membrane 208 may
be subjected to physiological mechanical strain generated by cyclic
stretching of the membrane 208 and/or the flow of biological fluids
(e.g. air, mucus, blood) to recapitulate the native
microenvironment of the alveoli and underlying pulmonary
capillaries. In an embodiment, the culture conditions of cells upon
the membrane 208 may be optimized under extracellular matrix (ECM)
coating, media perfusion, or cyclic mechanical strain to maintain
morphological and functional characteristics of the co-cultured
cells and to permit their direct cellular interaction across the
membrane 208. The device 200 would thus permit long-term cell
culture and dynamic mechanical stretching of a adjacent monolayers
of lung epithelial or endothelial cells grown on the membrane at
the same time.
[0112] In utilizing the membrane 208 in simulating the
tissue-tissue interface between the alveolar epithelium and
pulmonary endothelium in the lung, one method may be to apply type
I alveolar epithelial cells to the side of the membrane 208 facing
the first section 250A (hereinafter top side of membrane) to mimic
the epithelial layer. It is possible, however, to mix type I-like
and type II-like alveolar epithelial cells at a ratio of
approximately 7:13 to reconstruct the in vivo cellular composition
of the alveolar epithelium. In the example method, lung
microvascular endothelial cells are cultured on the opposite side
of the membrane 208 facing the second section 250B (hereinafter
bottom side of membrane). In the example method, negative pressure
is cyclically applied to the device 200 to cause the membrane 208
to continuously expand and contract along its plane.
[0113] During such operation, a physiological alveolar-capillary
unit may be formed on the membrane 208 since typical junctional
structures may form on the membrane 207 and fluids as well as ions
be transported across the membrane 208 between the first and second
sections 250A, 250B. The formation of tight junctions on the
membrane 208 may be evaluated using on-chip immunohistochemical
detection of tight junction proteins such as ZO-1 and occludin.
Additionally or alternately, the exclusion of fluorescently labeled
large molecules (e.g. dextrans of different weight) may be
quantitated to determine the permeability of the membrane and
optimize epithelial membrane barrier formation by varying culture
conditions. Additionally, histological, biochemical, and
microfluorimetric techniques may be employed to demonstrate
formation of a functional alveolar-capillary unit that reproduces
the key structural organization of its in vivo counterpart on the
membrane 208.
[0114] In an example, the gas exchange function of the
tissue-tissue interface self assembled on membrane 208 may be
determined by injecting different fluids, each having their own
oxygen partial pressures and blood, into the respective first and
second sections 250A, 250B, whereby the first section 250A acts as
the alveolar compartment and the second section 250B acts as the
microvascular compartment. A blood-gas measurement device
preferably within the device 200 is used to measure the level of
oxygen in the blood in the respective sections 250A, 250B before
and after the passing of the blood through the device. For example,
blood can flow through the channel 250B while air is being injected
into the upper channel 250A, whereby the exiting air is collected
and measured to determine the oxygen level using an oximeter.
Oximeters can be integrated with the existing system or as a
separate unit connected to the outlet port of one or more central
microchannels. In an embodiment, air or another medium with
aerosols containing drugs or particulates may flow through the
device, whereby the transport of these drugs or particulates to the
blood via the membrane is then measured. It is also contemplated
that pathogens or cytokines are added to the air or gaseous medium
side and then the sticking of immune cells to nearby capillary
endothelium and their passage along with edema fluid from the blood
side to the airway side, as well as pathogen entry into blood, are
measured.
[0115] Since the functionality of an epithelium requires
polarization of constituent cells, the structure of the membrane
may be visualized using transmission electron microscopy,
immunohistocytochemistry, confocal microscopy, or other appropriate
means to monitor the polarization of the alveolar epithelial cell
side of the membrane 208. In a lung mimic embodiment, a fluorescent
dye may be applied to the first and/or second microchannels 250A,
250B to determine pulmonary surfactant production by the airway
epithelium at the membrane 208. In particular, alveolar epithelial
cells on the membrane 208 can be monitored by measuring the
fluorescence resulting from cellular uptake of the fluorescence dye
that specifically labels intracellular storage of pulmonary
surfactant (e.g. quinacrine) or using specific antibodies.
[0116] One of the unique capabilities of the tissue interface
device 200 allows development of in vitro models that simulate
inflammatory responses of the lung at the organ level to allow
study of how immune cells migrate from the blood, through the
endothelium and into the alveolar compartment. One way this is
achieved is by controlled and programmable microfluidic delivery of
pro-inflammatory factors (e.g. IL-1.beta., TNF-.alpha., IL-8,
silica micro- and nanoparticles, pathogens) to the first section
250A as well as whole human blood flowing or medium containing
circulating immune cells in the second section 250B. Electrical
resistance and short circuit current across the membrane may be
monitored to study changes in the vascular permeability,
extravasation of fluid and cell passage into the alveolar space
during inflammatory responses. Fluorescence microscopy can be used
to visualize dynamic cell motile behavior during the extravasation
response.
[0117] The tissue interface device 200 may also be used to examine
how nanomaterials behave with respect to the lung tissue-tissue
interface. In particular, nanomaterials (e.g. silica nanoparticles,
superparamagnetic nanoparticles, gold nanoparticles, single-walled
carbon nanotubes) may be applied to the airway surface of the
membrane 208 to investigate potential toxic effects of
nanomaterials on airway or endothelial cells grown on the membrane
208, as well as their passage from the airway channel into the
blood channel. For instance, sensors 120 can be used to monitor
transmigration of nanomaterials through tissue barriers formed on
the membrane 208 and nanomaterial-induced changes in barrier
functions such as gas exchange and fluid/ion transport.
[0118] The tissue interface device 200 permits direct analysis of a
variety of important areas of lung biology and physiology including
but not limited to gas exchange, fluid/ion transport, inflammation,
infection, edema/respiratory distress syndrome, cancer and
metastasis development, fungal infection, drug delivery as well as
drug screening, biodetection, and pulmonary mechanotransduction. In
addition, the device 200 allows for accurately modeling biological
tissue-tissue interfaces found in other physiological systems such
as the blood-brain barrier, intestine, bone marrow, glomerulus, and
cancerous tumor microenvironment. As stated above, more than one
tissue interface device 200 may be multiplexed and automated to
provide high-throughput analysis of cell and tissue responses to
drugs, chemicals, particulates, toxins, pathogens or other
environmental stimuli for drug, toxin and vaccine screening, as
well as toxicology and biodetection applications. The device may be
used for studying complex tissue and organ physiology in vitro, as
well as tissue and organ engineering in vivo with biocompatible or
biodegradable devices.
[0119] In an embodiment, the tissue interface device 200 can be
used to produce artificial tissue layers therein. In the
embodiment, two or more different types of cells are applied on
opposing surfaces of the membrane 208 and grown under conditions
that mimic the appropriate physiological environments. Additionally
or alternatively, a pressure differential can be applied between
the central microchannel and at least one of the operating
microchannels which causes the microchannel walls to move and thus
causes the membrane 208 to undergo expansion/contraction along its
plane.
[0120] In another example, the device 200 utilizes the porous
membrane 208, whereby lung cells are grown on one side of the
membrane 208 and endothelial cells are maintained on the other side
of the membrane 208. During the operation of the device 200, these
two cells layers communicate with each other through passage of
chemical and molecular cues through the pores on the membrane 208.
This communication may be monitored and analyzed to understand how
the cells function differently as a tissue-tissue interface, with
or without physiological mechanical simulation, and compared to
when grown as single tissue types in isolation as in standard
tissue culture systems. By monitoring changes in cell and tissue
physiology, as well as passage of chemicals, molecules,
particulates and cells across this tissue-tissue interface,
information is obtained which may be used to produce more effective
drugs or therapies, to identify previously unknown toxicities, and
to significantly shorten the timescale of these development
processes. In particular, the behavior of cells in such a
controlled environment allows researchers to study a variety of
physiological phenomena taking place in the systems mentioned above
that can not be recreated using conventional in vitro culture
techniques. In other words, the device 200 functions to create a
monitorable artificial blood-air barrier outside a patient's body
and in a controllable environment that still retains key
physiological functions and structures of the lung. It should be
noted that although the device above is described in terms of
mimicking lung function, the device may easily be configured to
mimic other physiological systems such as peristalsis and
absorption in the gastrointestinal tract containing living
microbial populations, perfusion and urine production in the
kidney, function of the blood-brain barrier, effects of mechanical
deformation on skin aging, bone marrow-microvessel interface with
hematopoietic stem cell niche, and the like.
[0121] Details of membrane surface treatment and types of media
which can be applied to the membrane and/or through the central
microchannels 250A, 250B in operating the device will now be
discussed. The membrane including the porous membrane can be coated
with substances such as various cell adhesion promoting substances
or ECM proteins, such as fibronectin, laminin or various collagen
types or combinations thereof, as shown in FIG. 4D. In general, as
shown in FIG. 4D, one or more substances 608 is shown on one
surface of the membrane 604 whereas another substance 610 is
applied to the opposing surface of the membrane 604, or both
surfaces can be coated with the same substance. In some
embodiments, the ECMs, which may be ECMs produced by cells, such as
primary cells or embryonic stem cells, and other compositions of
matter are produced in a serum-free environment.
[0122] In an embodiment, one coats the membrane with a combination
of a cell adhesion factor and a positively-charged molecule that
are bound to the membrane to improve cell attachment and stabilize
cell growth. The positively charged molecule can be selected from
the group consisting of polylysine, chitosan, poly(ethyleneimine)
or acrylics polymerized from acrylamide or methacrylamide and
incorporating positively-charged groups in the form of primary,
secondary or tertiary amines, or quaternary salts. The cell
adhesion factor can be added to the membrane and is preferably
fibronectin, laminin, collagen, vitronectin or tenascin, or
fragments or analogs having a cell binding domain thereof. The
positively-charged molecule and the cell adhesion factor can be
covalently bound to the membrane. In another embodiment, the
positively-charged molecule and the cell adhesion factor are
covalently bound to one another and either the positively-charged
molecule or the cell adhesion factor is covalently bound to the
membrane. Also, the positively-charged molecule or the cell
adhesion factor or both cam be provided in the form of a stable
coating non-covalently bound to the membrane.
[0123] In an embodiment, the cell attachment-promoting substances,
matrix-forming formulations, and other compositions of matter are
sterilized to prevent unwanted contamination. Sterilization may be
accomplished, for example, by ultraviolet light, filtration, or
heat. Antibiotics may also be added, particularly during
incubation, to prevent the growth of bacteria, fungi and other
undesired micro-organisms. Such antibiotics include, by way of
non-limiting example, gentamicin, streptomycin, penicillin,
amphotericin and ciprofloxacin.
[0124] In another embodiment, the membrane is coated with cell
cultures, including without limitation, primary cell cultures,
established cell lines, or stem cell cultures, such as ESC,
attached to ECM substances that comprise or consist essentially of
fibronectin or collagen.
[0125] In an embodiment, the primary cells or cell lines attached
to the membrane may be alveolar cells, endothelial cells,
intestinal cells, keratinocytes, which include without limitation,
human dermal keratinocytes, or any other type of cell listed
elsewhere in this specification or well known to one skilled in the
art. In other embodiments, the primary cells may be fibroblast
cells, which include without limitation, human fetal fibroblast
cells. In some embodiments, the stem cells of the stem cell
cultures are embryonic stem cells. The source of embryonic stem
cells can include without limitation mammals, including non-human
primates and humans. Non-limiting examples of human embryonic stem
cells include lines BG01, BG02, BG03, BGO1v, CHA-hES-1, CHA-hES-2,
FCNCBS1, FCNCBS2, FCNCBS3, H1, H7, H9, H13, H14, HSF-1, H9.1, H9.2,
HES-1, HES-2, HES-3, HES-4, HES-5, HES-6, hES-1-2, hES-3-0,
hES-4-0, hES-5-1, hES-8-1, hES-8-2, hES-9-1, hES-9-2, hES-101,
hICM8, hICM9, hICM40, hICM41, hICM42, hICM43, HSF-6, HUES-1,
HUES-2, HUES-3, HUES-4 HUES-5, HUES-6, HUES-7 HUES-8, HUES-9,
HUES-10, HUES-11, HUES-12, HUES-13, HUES-14, HUESS-15, HUES-16,
HUES-17, 13, 14, 16, 13.2, 13.3, 16.2, J3, J3.2, MB01, MB02, MB03,
Miz-hES1, RCM-1, RLS ES 05, RLS ES 07, RLS ES 10, RLS ES 13, RLS ES
15, RLS ES 20, RLS ES 21, SA01, SA02, and SA03. In an embodiment,
the stem cells of the stem cell cultures are induced pluripotent
stem cells.
[0126] In an embodiment, the cell cultures may be cell cultures
such as primary cell cultures or stem cell cultures which are
serum-free. In some these embodiments, a serum-free primary cell
ECM is used in conjunction with a primary cell serum-free medium
(SFM). Suitable SFM include without limitation (a) EPILIFE.RTM.
Serum Free Culture Medium supplemented with EPILIFE.RTM. Defined
Growth Supplement and (b) Defined Keratinocyte-SFM supplemented
with Defined Keratinocyte-SFM Growth Supplement, all commercially
available from Gibco/Invitrogen (Carlsbad, Calif., US). In some of
these embodiments, a serum-free stem cell ECM is used in
conjunction with stem cell SFM. Suitable SFM include without
limitation STEMPRO.RTM. hESC Serum Free Media (SFM) supplemented
with basic fibroblast growth factor and .beta.-mercaptoethanol,
KNOCKOUT.TM.. D-MEM supplemented with KNOCKOUT.TM.. Serum
Replacement (SR), STEMPRO.RTM.. MSC SFM and STEMPRO.RTM.. NSC SFM,
all commercially available from Gibco/Invitrogen (Carlsbad, Calif.,
US).
[0127] In an embodiment, the compositions can also be xeno-free. A
composition of matter is said to be "xeno-free" when it is devoid
of substances from any animal other than the species of animal from
which the cells are derived. In an embodiment, the cell cultures
which may be cell cultures such as primary cell cultures or stem
cell cultures are xeno-free. In these embodiments, a xeno-free ECM
which may be an ECM such as a primary cell ECM or ECM designed
specifically to support stem cell growth or differentiation. These
matrices may be specifically designed to be xeno-free.
[0128] In an embodiment, the cell cultures are primary cells or
stem cells cultured in a conditioned culture medium. In other
embodiments, the culture medium is an unconditioned culture
medium.
[0129] In an embodiment, the cell culture conditions are completely
defined. In these embodiments, one uses a completely defined cell
culture medium in the fluid chambers. Suitable media include
without limitation, for primary cells, EPILIFE.RTM.. Serum Free
Culture Medium supplemented with EPILIFE.RTM.. Defined Growth
Supplement, and, for stem cells, STEMPRO.RTM.. hESC SFM, all
commercially available from Gibco/Invitrogen, Carlsbad, Calif.,
US.
[0130] To study the effects of pharmaceuticals, environmental
stressors, pathogens, toxins and such, one can add these into the
desired cell culture medium suitable for growing the cells attached
to the porous membrane in the channel. Thus, one can introduce
pathogens, such as bacteria, viruses, aerosols, various types of
nanoparticles, toxins, gaseous substances, and such into the
culture medium which flows in the chambers to feed the cells.
[0131] A skilled artisan will also be able to control the pH
balance of the medium according to the metabolic activity of the
cells to maintain the pH in a suitable level for any cell or tissue
type in question. Monitors and adjustment systems to monitor and
adjust pH may be inserted into the device.
[0132] The membrane is preferably coated on one or both sides with
cells, molecules or other matter, whereby the device provides a
controlled environment to monitor cell behavior along and/or
between the microchannels via the membrane. One can use any cells
from a multicellular organisms in the device. For example, human
body comprises at least 210 known types of cells. A skilled artisan
can easily construct useful combinations of the cells in the
device. Cell types (e.g., human) which can be used in the devices
include, but are not limited to cells of the integumentary system
including but not limited to Keratinizing epithelial cells,
Epidermal keratinocyte (differentiating epidermal cell), Epidermal
basal cell (stem cell), Keratinocyte of fingernails and toenails,
Nail bed basal cell (stem cell), Medullary hair shaft cell,
Cortical hair shaft cell, Cuticular hair shaft cell, Cuticular hair
root sheath cell, Hair root sheath cell of Huxley's layer, Hair
root sheath cell of Henle's layer, External hair root sheath cell,
Hair matrix cell (stem cell); Wet stratified barrier epithelial
cells, such as Surface epithelial cell of stratified squamous
epithelium of cornea, tongue, oral cavity, esophagus, anal canal,
distal urethra and vagina, basal cell (stem cell) of epithelia of
cornea, tongue, oral cavity, esophagus, anal canal, distal urethra
and vagina, Urinary epithelium cell (lining urinary bladder and
urinary ducts); Exocrine secretory epithelial cells, such as
Salivary gland mucous cell (polysaccharide-rich secretion),
Salivary gland serous cell (glycoprotein enzyme-rich secretion),
Von Ebner's gland cell in tongue (washes taste buds), Mammary gland
cell (milk secretion), Lacrimal gland cell (tear secretion),
Ceruminous gland cell in ear (wax secretion), Eccrine sweat gland
dark cell (glycoprotein secretion), Eccrine sweat gland clear cell
(small molecule secretion), Apocrine sweat gland cell (odoriferous
secretion, sex-hormone sensitive), Gland of Moll cell in eyelid
(specialized sweat gland), Sebaceous gland cell (lipid-rich sebum
secretion), Bowman's gland cell in nose (washes olfactory
epithelium), Brunner's gland cell in duodenum (enzymes and alkaline
mucus), Seminal vesicle cell (secretes seminal fluid components,
including fructose for swimming sperm), Prostate gland cell
(secretes seminal fluid components), Bulbourethral gland cell
(mucus secretion), Bartholin's gland cell (vaginal lubricant
secretion), Gland of Littre cell (mucus secretion), Uterus
endometrium cell (carbohydrate secretion), Isolated goblet cell of
respiratory and digestive tracts (mucus secretion), Stomach lining
mucous cell (mucus secretion), Gastric gland zymogenic cell
(pepsinogen secretion), Gastric gland oxyntic cell (hydrochloric
acid secretion), Pancreatic acinar cell (bicarbonate and digestive
enzyme secretion), pancreatic endocrine cells, Paneth cell of small
intestine (lysozyme secretion), intestinal epithelial cells, Types
I and II pneumocytes of lung (surfactant secretion), and/or Clara
cell of lung.
[0133] One can also coat the membrane with Hormone secreting cells,
such as endocrine cells of the islet of Langerhands of the
pancreas, Anterior pituitary cells, Somatotropes, Lactotropes,
Thyrotropes, Gonadotropes, Corticotropes, Intermediate pituitary
cell, secreting melanocyte-stimulating hormone; and Magnocellular
neurosecretory cells secreting oxytocin or vasopressin; Gut and
respiratory tract cells secreting serotonin, endorphin,
somatostatin, gastrin, secretin, cholecystokinin, insulin,
glucagon, bombesin; Thyroid gland cells such as thyroid epithelial
cell, parafollicular cell, Parathyroid gland cells, Parathyroid
chief cell, Oxyphil cell, Adrenal gland cells, chromaffin cells
secreting steroid hormones (mineral corticoids and gluco
corticoids), Leydig cell of testes secreting testosterone, Theca
interna cell of ovarian follicle secreting estrogen, Corpus luteum
cell of ruptured ovarian follicle secreting progesterone, Granulosa
lutein cells, Theca lutein cells, Juxtaglomerular cell (renin
secretion), Macula densa cell of kidney, Peripolar cell of kidney,
and/or Mesangial cell of kidney.
[0134] Additionally or alternatively, one can treat at least one
side of the membrane with Metabolism and storage cells such as
Hepatocyte (liver cell), White fat cell, Brown fat cell, Liver
lipocyte. One can also use Barrier function cells (Lung, Gut,
Exocrine Glands and Urogenital Tract) or Kidney cells such as
Kidney glomerulus parietal cell, Kidney glomerulus podocyte, Kidney
proximal tubule brush border cell, Loop of Henle thin segment cell,
Kidney distal tubule cell, and/or Kidney collecting duct cell.
[0135] Other cells that can be used in the device include Type I
pneumocyte (lining air space of lung), Pancreatic duct cell
(centroacinar cell), Nonstriated duct cell (of sweat gland,
salivary gland, mammary gland, etc.), principal cell, Intercalated
cell, Duct cell (of seminal vesicle, prostate gland, etc.),
Intestinal brush border cell (with microvilli), Exocrine gland
striated duct cell, Gall bladder epithelial cell, Ductulus efferens
nonciliated cell, Epididymal principal cell, and/or Epididymal
basal cell.
[0136] One can also use Epithelial cells lining closed internal
body cavities such as Blood vessel and lymphatic vascular
endothelial fenestrated cell, Blood vessel and lymphatic vascular
endothelial continuous cell, Blood vessel and lymphatic vascular
endothelial splenic cell, Synovial cell (lining joint cavities,
hyaluronic acid secretion), Serosal cell (lining peritoneal,
pleural, and pericardial cavities), Squamous cell (lining
perilymphatic space of ear), Squamous cell (lining endolymphatic
space of ear), Columnar cell of endolymphatic sac with microvilli
(lining endolymphatic space of ear), Columnar cell of endolymphatic
sac without microvilli (lining endolymphatic space of ear), Dark
cell (lining endolymphatic space of ear), Vestibular membrane cell
(lining endolymphatic space of ear), Stria vascularis basal cell
(lining endolymphatic space of ear), Stria vascularis marginal cell
(lining endolymphatic space of ear), Cell of Claudius (lining
endolymphatic space of ear), Cell of Boettcher (lining
endolymphatic space of ear), Choroid plexus cell (cerebrospinal
fluid secretion), Pia-arachnoid squamous cell, Pigmented ciliary
epithelium cell of eye, Nonpigmented ciliary epithelium cell of
eye, and/or Corneal endothelial cell.
[0137] The following cells can be used in the device by adding them
to the surface of the membrane in culture medium. These cells
include cells such as Ciliated cells with propulsive function such
as Respiratory tract ciliated cell, Oviduct ciliated cell (in
female), Uterine endometrial ciliated cell (in female), Rete testis
ciliated cell (in male), Ductulus efferens ciliated cell (in male),
and/or Ciliated ependymal cell of central nervous system (lining
brain cavities).
[0138] One can also plate cells that secrete specialized ECMs, such
as Ameloblast epithelial cell (tooth enamel secretion), Planum
semilunatum epithelial cell of vestibular apparatus of ear
(proteoglycan secretion), Organ of Corti interdental epithelial
cell (secreting tectorial membrane covering hair cells), Loose
connective tissue fibroblasts, Corneal fibroblasts (corneal
keratocytes), Tendon fibroblasts, Bone marrow reticular tissue
fibroblasts, Other nonepithelial fibroblasts, Pericyte, Nucleus
pulposus cell of intervertebral disc, Cementoblast/cementocyte
(tooth root bonelike cementum secretion), Odontoblast/odontocyte
(tooth dentin secretion), Hyaline cartilage chondrocyte,
Fibrocartilage chondrocyte, Elastic cartilage chondrocyte,
Osteoblast/osteocyte, Osteoprogenitor cell (stem cell of
osteoblasts), Hyalocyte of vitreous body of eye, Stellate cell of
perilymphatic space of ear, Hepatic stellate cell (Ito cell),
and/or Pancreatic stellate cell.
[0139] Additionally or alternatively, contractile cells, such as
Skeletal muscle cells, Red skeletal muscle cell (slow), White
skeletal muscle cell (fast), Intermediate skeletal muscle cell,
nuclear bag cell of muscle spindle, nuclear chain cell of muscle
spindle, Satellite cell (stem cell), Heart muscle cells, Ordinary
heart muscle cell, Nodal heart muscle cell, Purkinje fiber cell,
Smooth muscle cell (various types), Myoepithelial cell of iris,
Myoepithelial cell of exocrine glands can be used in the present
device.
[0140] The following cells can also be used in the present device:
Blood and immune system cells, such as Erythrocyte (red blood
cell), Megakaryocyte (platelet precursor), Monocyte, Connective
tissue macrophage (various types), Epidermal Langerhans cell,
Osteoclast (in bone), Dendritic cell (in lymphoid tissues),
Microglial cell (in central nervous system), Neutrophil
granulocyte, Eosinophil granulocyte, Basophil granulocyte, Mast
cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, Natural
Killer T cell, B cell, Natural killer cell, Reticulocyte, Stem
cells and committed progenitors for the blood and immune system
(various types). One can use these cells as single cell types or in
mixtures to determine effects of the immune cells in the tissue
culture system.
[0141] One can also treat the membranes with one or more Nervous
system cells, Sensory transducer cells such as Auditory inner hair
cell of organ of Corti, Auditory outer hair cell of organ of Corti,
Basal cell of olfactory epithelium (stem cell for olfactory
neurons), Cold-sensitive primary sensory neurons, Heat-sensitive
primary sensory neurons, Merkel cell of epidermis (touch sensor),
Olfactory receptor neuron, Pain-sensitive primary sensory neurons
(various types); Photoreceptor cells of retina in eye including
Photoreceptor rod cells, Photoreceptor blue-sensitive cone cell of
eye, Photoreceptor green-sensitive cone cell of eye, Photoreceptor
red-sensitive cone cell of eye, Proprioceptive primary sensory
neurons (various types); Touch-sensitive primary sensory neurons
(various types); Type I carotid body cell (blood pH sensor); Type
II carotid body cell (blood pH sensor); Type I hair cell of
vestibular apparatus of ear (acceleration and gravity); Type II
hair cell of vestibular apparatus of ear (acceleration and
gravity); and/or Type I taste bud cell.
[0142] One can further use Autonomic neuron cells such as
Cholinergic neural cell (various types), Adrenergic neural cell
(various types), Peptidergic neural cell (various types) in the
present device. Further, sense organ and peripheral neuron
supporting cells can also be used. These include, for example,
Inner pillar cell of organ of Corti, Outer pillar cell of organ of
Corti, Inner phalangeal cell of organ of Corti, Outer phalangeal
cell of organ of Corti, Border cell of organ of Corti, Hensen cell
of organ of Corti, Vestibular apparatus supporting cell, Type I
taste bud supporting cell, Olfactory epithelium supporting cell,
Schwann cell, Satellite cell (encapsulating peripheral nerve cell
bodies) and/or Enteric glial cell. In some embodiments, one can
also use central nervous system neurons and glial cells such as
Astrocyte (various types), Neuron cells (large variety of types,
still poorly classified), Oligodendrocyte, and Spindle neuron.
[0143] Lens cells such as Anterior lens epithelial cell and
Crystallin-containing lens fiber cell can also be used in the
present device. Additionally, one can use pigment cells such as
melanocytes and retinal pigmented epithelial cells; and germ cells,
such as Oogonium/Oocyte, Spermatid, Spermatocyte, Spermatogonium
cell (stem cell for spermatocyte), and Spermatozoon.
[0144] In some embodiments one can add to the membrane nurse cells
Ovarian follicle cell, Sertoli cell (in testis), Thymus epithelial
cell. One can also use interstitial cells such as interstitial
kidney cells.
[0145] In an embodiment, one can coat at least one side of the
membrane with epithelial cells. Epithelium is a tissue composed of
cells that line the cavities and surfaces of structures throughout
the body. Many glands are also formed from epithelial tissue. It
lies on top of connective tissue, and the two layers are separated
by a basement membrane. In humans, epithelium is classified as a
primary body tissue, the other ones being connective tissue, muscle
tissue and nervous tissue. Epithelium is often defined by the
expression of the adhesion molecule e-cadherin (as opposed to
n-cadherin, which is used by neurons and cells of the connective
tissue).
[0146] Functions of epithelial cells include secretion, selective
absorption, protection, transcellular transport and detection of
sensation and they commonly as a result present extensive
apical-basolateral polarity (e.g. different membrane proteins
expressed) and specialization. Examples of epithelial cells include
squamous cells that have the appearance of thin, flat plates. They
fit closely together in tissues; providing a smooth, low-friction
surface over which fluids can move easily. The shape of the nucleus
usually corresponds to the cell form and helps to identify the type
of epithelium. Squamous cells tend to have horizontally flattened,
elliptical nuclei because of the thin flattened form of the cell.
Classically, squamous epithelia are found lining surfaces utilizing
simple passive diffusion such as the alveolar epithelium in the
lungs. Specialized squamous epithelia also form the lining of
cavities such as the blood vessels (endothelium) and heart
(mesothelium) and the major cavities found within the body.
[0147] Another example of epithelial cells is cuboidal cells.
Cuboidal cells are roughly cuboidal in shape, appearing square in
cross section. Each cell has a spherical nucleus in the centre.
Cuboidal epithelium is commonly found in secretive or absorptive
tissue: for example the (secretive) exocrine gland the pancreas and
the (absorptive) lining of the kidney tubules as well as in the
ducts of the glands. They also constitute the germinal epithelium
which produces the egg cells in the female ovary and the sperm
cells in the male testes.
[0148] Yet another type of epithelial cells are columnar epithelial
cells that are elongated and column-shaped. Their nuclei are
elongated and are usually located near the base of the cells.
Columnar epithelium forms the lining of the stomach and intestines.
Some columnar cells are specialised for sensory reception such as
in the nose, ears and the taste buds of the tongue. Goblet cells
(unicellular glands) are found between the columnar epithelial
cells of the duodenum. They secrete mucus, which acts as a
lubricant.
[0149] Still another example of the epithelial cells are
pseudostratified cells. These are simple columnar epithelial cells
whose nuclei appear at different heights, giving the misleading
(hence "pseudo") impression that the epithelium is stratified when
the cells are viewed in cross section. Pseudostratified epithelium
can also possess fine hair-like extensions of their apical
(luminal) membrane called cilia. In this case, the epithelium is
described as "ciliated" pseudostratified epithelium. Cilia are
capable of energy dependent pulsatile beating in a certain
direction through interaction of cytoskeletal microtubules and
connecting structural proteins and enzymes. The wafting effect
produced causes mucus secreted locally by the goblet cells (to
lubricate and to trap pathogens and particles) to flow in that
direction (typically out of the body). Ciliated epithelium is found
in the airways (nose, bronchi), but is also found in the uterus and
Fallopian tubes of females, where the cilia propel the ovum to the
uterus.
[0150] Epithelium lines both the outside (skin) and the inside
cavities and lumen of bodies. The outermost layer of our skin is
composed of dead stratified squamous, keratinised epithelial
cells.
[0151] Tissues that line the inside of the mouth, the oesophagus
and part of the rectum are composed of nonkeratinized stratified
squamous epithelium. Other surfaces that separate body cavities
from the outside environment are lined by simple squamous,
columnar, or pseudostratified epithelial cells. Other epithelial
cells line the insides of the lungs, the gastrointestinal tract,
the reproductive and urinary tracts, and make up the exocrine and
endocrine glands. The outer surface of the cornea is covered with
fast-growing, easily-regenerated epithelial cells. Endothelium (the
inner lining of blood vessels, the heart, and lymphatic vessels) is
a specialized form of epithelium. Another type, mesothelium, forms
the walls of the pericardium, pleurae, and peritoneum.
[0152] Accordingly, one can recreate any of these tissues in the
cell culture device as described by plating applicable cell types
on the porous membranes and applying applicable vacuum to provide
physiological or artificial mechanical force on the cells to mimic
physiological forces, such as tension on skin or mechanical strain
on lung. In an embodiment, one side of the membrane is coated with
epithelial cells and the other side is coated with endothelial
cells.
[0153] The endothelium is the thin layer of cells that line the
interior surface of blood vessels, forming an interface between
circulating blood in the lumen and the rest of the vessel wall.
Endothelial cells line the entire circulatory system, from the
heart to the smallest capillary. These cells reduce turbulence of
the flow of blood allowing the fluid to be pumped farther.
Endothelial tissue is a specialized type of epithelium tissue (one
of the four types of biological tissue in animals). More
specifically, it is simple squamous epithelium.
[0154] The foundational model of anatomy makes a distinction
between endothelial cells and epithelial cells on the basis of
which tissues they develop from and states that the presence of
vimentin rather than keratin filaments separate these from
epithelial cells. Endothelium of the interior surfaces of the heart
chambers are called endocardium. Both blood and lymphatic
capillaries are composed of a single layer of endothelial cells
called a monolayer. Endothelial cells are involved in many aspects
of vascular biology, including: vasoconstriction and vasodilation,
and hence the control of blood pressure; blood clotting (thrombosis
& fibrinolysis); atherosclerosis; formation of new blood
vessels (angiogenesis); inflammation and barrier function--the
endothelium acts as a selective barrier between the vessel lumen
and surrounding tissue, controlling the passage of materials and
the transit of white blood cells into and out of the bloodstream.
Excessive or prolonged increases in permeability of the endothelial
monolayer, as in cases of chronic inflammation, may lead to tissue
oedema/swelling. In some organs, there are highly differentiated
endothelial cells to perform specialized `filtering` functions.
Examples of such unique endothelial structures include the renal
glomerulus and the blood-brain barrier.
[0155] In an embodiment, the membrane side that contains cultured
endothelial cells can be exposed to various test substances and
also white blood cells or specific immune system cells to study
effects of the test agents on the function of the immune system
cells at the tissue level.
[0156] Details on how the tissue interface device 200 is formed
will now be discussed in accordance with an embodiment. The
fabrication of the PDMS membrane preferably involves parallel
processing of multiple parts which are assembled in stages. FIG. 4A
illustrates a perspective view of a master 600 in accordance with
an embodiment which is ultimately used to produce the porous
membrane 208. As shown in FIG. 4A, the master 600 is preferably
formed by patterning a photoresist to the desired shape and size on
a silicon substrate.
[0157] It should be noted that the posts 602 may be designed in any
desired array depending on the intended design of the membrane 208.
For example, the posts 602 may be arranged in a circular pattern to
correspondingly form a circular patterned set of pores in the
membrane 208. It should be noted that the posts 602 may have any
other cross sectional shape other than pentagonal to make the
corresponding pores in the membrane, as discussed above. It should
also be noted that the master 600 may contain different height
ridges to create non planar membranes.
[0158] Thereafter, as shown in FIG. 4B, the master 600 is
preferably spin-coated with PDMS to form a spin coated layer 604.
Thereafter, the spin-coated layer 604 is cured for a set time and
temperature (e.g. 110.degree. C. at 15 minutes) and peeled off the
master 600 to produce a thin PDMS membrane 604 having the array of
pentagonal through-holes 606, as shown in FIG. 4C. The example
shown depicts fabrication of a 10 .mu.m-thick PDMS membrane,
although other thickness values are contemplated.
[0159] Although other materials may be used, PDMS has useful
properties in biology in that it is a moderately stiff elastomer (1
MPa) which is non-toxic and is optically transparent to 300 nm.
PDMS is intrinsically very hydrophobic, but can be converted to
hydrophilic form by treatment with plasma. The membrane 604 may be
engineered for a variety of purposes, some discussed above. For
example, the pores 606 on the membrane 604 may be coated or filled
with ECM molecules or gels, such as MATRIGEL, laminin, collagen,
fibronectin, fibrin, elastin, etc., which are known to those
skilled in the art. The tissue-tissue interface may be coated by
culturing different types of cells on each side of the membrane
604, as shown in FIG. 4D. In particular, as shown in FIG. 4D, one
type of cells 608 are coated on one side of the membrane 604
whereas another type of cells 610 are coated on the opposing side
of the membrane 604.
[0160] FIGS. 5A and 5B illustrate the process how the first outer
body portion 202, a second outer body portion 204 are formed in
accordance with an embodiment. The first and second outer body
portions 202, 204 are preferably formed using soft lithography
techniques, although other techniques well known in the art are
contemplated. In an embodiment, a photoresist (not shown) is formed
on a substrate in which the photoresist has positive relief
features which mirror the desired branching configuration in the
first outer body portion. Similarly, a second photoresist (not
shown) is formed on another substrate in which the second
photoresist has corresponding positive relief features which mirror
the branching configuration in the second outer body portion 204.
The microchannels along with the communicating ports and port
apertures are preferably generated by preferably casting PDMS or
other appropriate material onto each master. Once the first and
second outer body portions 202, 204 are formed, through-holes which
serve as the port apertures are made through the PDMS slab
preferably using an aperture forming mechanism or stamp.
[0161] As shown in FIG. 5C, the already formed PDMS membrane 208 is
then sandwiched between the first outer body portion 202 and the
second outer body portion 204, whereby the microchannel walls 234,
244 as well as the outside walls 238, 248 are aligned using
appropriate manufacturing equipment and techniques. Thereafter, the
microchannel walls 234, 244 and outside walls are preferably bonded
to the membrane 208 using an appropriate adhesive or epoxy.
Additionally, the remaining portions of the outer body portions
202, 204 are permanently bonded to one another using an appropriate
adhesive or epoxy to form the overall device.
[0162] Subsequently, as shown in FIG. 5D, a PDMS etching solution
is introduced into the operating channels to etch away the PDMS
membrane segments in the operating channels. This results in
resulting in the generation of the two side operating channels 252
being free from the membrane, although the membrane is maintained
in the central microchannel, as shown in FIG. 5E. The above is
preferably formed using soft lithography techniques, the details of
which are described in "Soft Lithography in Biology and
Biochemistry," by Whitesides, et al., published Annual Review,
Biomed Engineering, 3.335-3.373 (2001), as well as "An Ultra-Thin
PDMS Membrane As A Bio/Micro-Nano Interface: Fabrication And
Characterization", by Thangawng et al., Biomed Microdevices, vol.
9, num. 4, 2007, p. 587-95, both of which are hereby incorporated
by reference.
[0163] FIG. 6 illustrates a schematic of a system having multiple
tissue interface devices in accordance with an embodiment. In
particular, as shown in FIG. 6, the system 700 includes one or more
CPUs 702 coupled to one or more fluid sources 704 and pressure
sources (not shown), whereby the preceding are coupled to three
shown tissue interface devices 706A, 706B, and 706C. It should be
noted that although three devices 706 are shown in this embodiment,
fewer or greater than three devices 706 are contemplated. In the
system 700, two of the three devices (i.e. 706A and 706B) are
connected in parallel with respect to the fluid source 704 and
devices 706A and 706C are connected in serial fashion with respect
to the fluid source 704. It should be noted that the shown
configuration is only one example and any other types of connection
patterns may be utilized depending on the application.
[0164] In the example shown, fluid from the fluid source 704 is
provided directly to the fluid inlets of devices 706A and 706B. As
the fluid passes through device 706A, it is output directly into
the fluid inlet port of devices 706B and 706C. Additionally, the
fluid outlet from device 706B is combined with the output from
device 706A into device 706C. With multiple devices operating, it
is possible to monitor, using sensor data, how the cells in the
fluid or membrane behave after the fluid has been passed through
another controlled environment. This system thus allows multiple
independent "stages" to be set up, where cell behavior in each
stage may be monitored under simulated physiological conditions and
controlled using the devices 706. One or more devices are connected
serially may provide use in studying chemical communication between
cells. For example, one cell type may secrete protein A in response
to being exposed to a particular fluid, whereby the fluid,
containing the secreted protein A, exits one device and then is
exposed to another cell type specifically patterned in another
device, whereby the interaction of the fluid with protein A with
the other cells in the other device can be monitored (e.g.
paracrine signaling). For the parallel configuration, one or more
devices connected in parallel may be advantageous in increasing the
efficiency of analyzing cell behavior across multiple devices at
once instead of analyzing the cell behavior through individual
devices separately.
[0165] FIG. 7A illustrates a perspective view of an organ mimic
device in accordance with an embodiment that contains three
parallel microchannels separated by two porous membranes. As shown
in FIG. 7A, the organ mimic device 800 includes operating
microchannels 802 and an overall central microchannel 804
positioned between the operating microchannels 802. The overall
central microchannel 804 includes multiple membranes 806A, 806B
positioned along respective parallel x-y planes which separate the
microchannel 804 into three distinct central microchannels 804A,
804B and 804C. The membranes 806A and 806B may be porous, elastic,
or a combination thereof. Positive and/or negative pressurized
media may be applied via operating channels 802 to create a
pressure differential to thereby cause the membranes 806A, 806B to
expand and contract along their respective planes in parallel.
[0166] FIG. 7B illustrates a perspective view of an organ mimic
device in accordance with an embodiment. As shown in FIG. 7B, the
tissue interface device 900 includes operating microchannels 902A,
902B and a central microchannel 904 positioned between the
microchannels 902. The central microchannel 904 includes multiple
membranes 906A, 906B positioned along respective parallel x-y
planes. Additionally, a wall 910 separates the central microchannel
into two distinct central microchannels, having respective
sections, whereby the wall 910 along with membranes 904A and 904B
define microchannels 904A, 904B, 904C, and 904D. The membranes 906A
and 906B at least partially porous, elastic or a combination
thereof.
[0167] The device in FIG. 7B differs from that in FIG. 7A in that
the operating microchannels 902A and 902B are separated by a wall
908, whereby separate pressures applied to the microchannels 902A
and 902B cause their respective membranes 904A and 904B to expand
or contract. In particular, a positive and/or negative pressure may
be applied via operating microchannels 902A to cause the membrane
906A to expand and contract along its plane while a different
positive and/or negative pressure is applied via operating
microchannels 902B to cause the membrane 906B to expand and
contract along its plane at a different frequency and/or magnitude.
Of course, one set of operating microchannels may experience the
pressure while the other set does not experience a pressure,
thereby only causing one membrane to actuate. It should be noted
that although two membranes are shown in the devices 800 and 900,
more than two membranes are contemplated and can be configured in
the devices.
[0168] In an example, shown in FIG. 7C, the device containing three
channels described in FIG. 7A has two membranes 806A and 806B which
are coated to determine cell behavior of a vascularized tumor. In
particular, membrane 806A is coated with a lymphatic endothelium on
its upper surface 805A and with stromal cells on its lower surface,
and stromal cells are also coated on the upper surface of the
second porous membrane 805B and a vascular endothelium on its
bottom surface 805C. Tumor cells are placed in the central
microchannel surrounded on top and bottom by layers of stromal
cells on the surfaces of the upper and lower membranes in section
804B. Fluids such as cell culture medium or blood enters the
vascular channel in section 804 C (you are missing a label 804C in
the diagram). Fluid such as cell culture medium or lymph enters the
lymphatic channel in section 804A. This configuration of the device
800 allows researchers to mimic and study tumor growth and invasion
into blood and lymphatic vessels during cancer metastasis. In the
example, one or more of the membranes 806A, 806B may
expand/contract in response to pressure through the operating
microchannels. Additionally or alternatively, the membranes may not
actuate, but may be porous or have grooves to allow cells to pass
through the membranes.
[0169] The unique capabilities of the present device have been
monitored in experiments that address acute toxicity and
extrapulmonary translocation of engineered nanomaterials induced by
physiological mechanical forces. The device has been used to model
pulmonary inflammation in which it can precisely recreate and
directly visualize the complex interplay of pulmonary tissues with
cytokines and blood-borne immune cells that transmigrate across the
alveolar-capillary barrier. Using this model, the device reveals
significant inflammatory responses of the mimicked lung to
nanomaterials. Finally, the device is used to simulate pulmonary
infection with bacteria and its clearance by neutrophil recruitment
and phagocytosis.
[0170] The device has been used in experiments which have led to
the discovery that physiological mechanical forces can induce or
exacerbate toxicity of engineered nanomaterials in the lung and may
facilitate their translocation into the systemic circulation.
Furthermore, in vitro models that simulate lung inflammation have
been developed that enable direct observation of the adhesion of
circulating blood-borne immune cells to inflamed endothelia and
their transmigration across the alveolar-capillary barrier. Based
on this model, significant proinflammatory activities of engineered
nanoparticles have been revealed. Based on this evidence, a model
of pulmonary infection can be established and re-creation may be
done of the innate immune response of the lung to bacteria mediated
by neutrophil infiltration into the alveoli and bacterial
phagocytosis.
[0171] The present device was utilized in several experiments,
whereby the device was used to mimic the living lung. The
observations and findings with the present device are described
hereafter. During normal inspiration of a real lung, the thoracic
cavity enlarges due to the contraction of the diaphragm and
expansion of the rib-cage and, as a result, the intrapleural
pressure outside the alveoli decreases. The increased pressure
difference across the alveolar wall causes the alveoli to expand
and forces air into the lungs, resulting in stretching of the
alveolar epithelium and endothelium in the surrounding capillaries.
Alveolar epithelial cells are co-cultured with pulmonary
microvascular endothelial cells on a thin porous membrane to
produce two opposing tissue layers that mimic the interface between
the alveolar epithelium and pulmonary endothelium. The
compartmentalized microchannel configuration makes it readily
possible to manipulate fluidic environment of the epithelium and
endothelium independently, and to apply physiological mechanical
strain.
[0172] In the experiment, co-culture of alveolar epithelial cells
and primary pulmonary microvascular endothelial cells of human
origin was developed over two weeks without loss of viability. The
microfluidic culture resulted in the production of tight
alveolar-capillary barriers with structural integrity as evidenced
by typical junctional complexes present in both epithelial and
endothelial layers. The microfluidic device was integrated with
computer-controlled vacuum to enable cyclic membrane/cell
stretching at varying frequencies and levels of strain in a
programmable manner. It was observed that applied vacuum generated
unidirectional tension which is uniform across the wide central
microchannel. Concurrently, it was discovered that this tension was
perceived by adherent cells and caused them to stretch and increase
their projected surface area. Also effective application of
mechanical strain to cells was confirmed by showing stretch-induced
alignment and transient calcium responses of endothelial cells.
[0173] Based on the unique capabilities afforded by on-chip
production of pulmonary tissues and faithful recapitulation of
their native microenvironment, the device was used to assess the
potential adverse effects of nanomaterials. Despite the widespread
use of engineered nanomaterials, much remains to be learned about
their risks to health and environment. Existing toxicology methods
rely on oversimplified in vitro models or lengthy, expensive animal
testing that often poses challenges to mechanistic studies at the
cellular level. To bridge the gap between cell culture studies and
animal models, the device was used to permit a more realistic,
accurate evaluation of nanomaterial toxicity in a tightly
controlled biomimetic microenvironment.
[0174] In the experiment, the alveolar epithelial tissues prepared
in the device were exposed to various nanomaterials and oxidative
stress was examined by measuring intracellular production of
reactive oxygen species (ROS) using microfluorimetry. Through the
testing of colloidal silica nanoparticles and quantum dots, it was
discovered that physiological mechanical strain can dramatically
increase nanoparticle-generated oxidative stress and induce early
toxic responses in the pulmonary epithelium. For example, when the
cells were exposed to 12 nm silica nanoparticles in combination
with a cyclic stretch of 10% strain at 0.2 Hz which simulates
normal respiration, ROS production increased by more than five
times after two hours, whereas nanoparticles or mechanical strain
alone did not cause any measurable responses over the duration of
the experiments (see FIG. 8). The response of cells treated with
carboxylated quantum dots showed similar trends (see FIG. 9). It
was noted that similar levels of ROS increase were achieved after
24 hour-long exposures to silica nanoparticles alone, as shown in
FIG. 9.
[0175] It was also found that cyclic strain alone did not have any
significant impact regardless of its duration, as shown in FIG. 9.
Taken together, these observations suggest that physiological
forces act in synergy with nanoparticles to exert early toxic
effects or aggravate nanoparticle toxicity in the lung. This
stretch-induced ROS response to nanomaterials depended on the level
of strain and induced apoptosis of the epithelial cells as detected
by caspase activity. When treated with a clinically used free
radical scavenger, N-acetylcysteine (NAC) during nanoparticle
exposure, the cells were completely rescued from oxidative stress
presumably due to the antioxidant activity of NAC leading to
increased intracellular glutathione. It was also observed that
oxidative stress generated by the combined effect of nanomaterials
and strain varied significantly with the type of nanomaterials. For
example, exposures to 50 nm superparamagnetic iron nanoparticles
under the same conditions only resulted in a transient increase in
oxidative stress. This unique ROS response was not observed in the
testing of other nanomaterials including single walled carbon
nanotubes, gold nanoparticles, polystyrene nanoparticles, and
quantum dots coated with polyethylene glycol, as shown below in
Table 1.
TABLE-US-00001 TABLE 1 ROS response ROS response Nanomaterials
Surface coating Size (0% strain) (10% strain) Polystyrene Carboxyl
groups 500 nm No No nanoparticles Carboxyl groups 200 nm No No
Amine groups 200 nm No No Carboxyl groups 100 nm No No Carboxyl
groups 20 nm No No Quantum dots Carboxyl groups 16 nm No Yes
polyethylene glycol 13 nm No No Silica N/A 12 nm No Yes
nanoparticles Magnetic iron Carboxyl groups 50 nm No Yes
nanoparticles Gold nanoparticles N/A 3 nm No No
[0176] To understand the influence of physiological forces on
tissue-nanomaterial interactions, confocal microscopy was used to
analyze internalization of 100 nm fluorescent nanoparticles into
the epithelial cells after 1 hour of exposure. However, the number
of particles or their aggregates detected in intracellular
compartments was much greater in the presence of mechanical strain,
and over 80% of the cells were found to have taken up the
nanoparticles, whereas the extent of nanoparticle uptake was
considerably smaller in the absence of strain. These results
indicate that physiological mechanical forces may facilitate
cellular uptake of nanomaterials, allowing them to interact with
subcellular components and thereby rendering them potentially more
harmful.
[0177] Moreover, the device provides an opportunity to investigate
extrapulmonary translocation of nanomaterials from the alveolar
space to the microvasculature. Increasing in vivo evidence suggests
that nanomaterials in the alveoli have the capacity to cross the
alveolar-capillary barrier and enter the pulmonary circulation,
potentially impacting other organs. To investigate this situation,
20 nm fluorescent nanoparticles were introduced on the epithelial
side and nanoparticle translocation was monitored by counting the
number of particles carried out of the lower vascular channel by
continuous fluid flow. This model revealed a marked increase in the
rate of nanoparticle migration into the vascular compartment under
physiological conditions with 10% cyclic strain, as compared to
transport across a relaxed, static tissue barrier. These findings
provide in vitro evidence that the inherent mechanical activity of
the living lung may allow nanomaterials to translocate from the
alveolar space into the bloodstream. The data from the experiment
also supports the systematic distribution and accumulation of
inhaled nanomaterials observed in animal studies and may
potentially contribute to delineating the mechanism of this
process, as well as providing a surrogate model system for studying
this response.
[0178] To further demonstrate the device's capabilities to
reconstitute the integrated organ-level responses in the lung, a
more sophisticated model was developed that incorporated
circulating blood-borne immune cells and reproduced the key steps
of lung inflammation. Generally, inflammatory responses in the lung
involve a highly coordinated multistep cascade of epithelial
production and release of early response cytokines, activation of
vascular endothelium through upregulation of leukocyte adhesion
molecules and subsequent leukocyte infiltration from the pulmonary
microcirculation into the alveolar space. To simulate this process,
the apical surface of the alveolar epithelium was first stimulated
with tumor necrosis factor-.alpha. (TNF-.alpha.), which is a potent
pro-inflammatory mediator, and endothelial activation was examined
by measuring the expression of intercellular adhesion molecule-1
(ICAM-1). In response to TNF-.alpha. stimulation of the alveolar
tissue for 5 hours, the endothelial cells on the opposite side of
the membrane dramatically increased their surface expression of
ICAM-1. Furthermore, the activated endothelium supported capture
and firm adhesion of human neutrophils flowing in the vascular
microchannel, which did not adhere in the absence of cytokine
exposure. Treatment of the epithelial cells with low doses of
TNF-.alpha. resulted in weak activation of the endothelium, which
caused captured neutrophils to roll continuously in the direction
of flow without being arrested. Direct microscopic visualization
revealed that adherent neutrophils became flattened and crawled
from a site of firm adhesion to distant locations where they
extravasated through the endothelium and transmigrated across the
alveolar-capillary barrier through the membrane pores over the
period of several minutes. The transmigrated neutrophils then
emigrated onto the apical surface of the alveolar epithelium
preferentially through paracellular junctions and were retained on
the epithelial layer in spite of fluid flow and cyclic stretching.
These sequential events successfully replicate the entire process
of neutrophil recruitment from the microvasculature to the alveolar
compartment, which is a hallmark of lung inflammation.
[0179] Using the device, proinflammatory effects of colloidal
silica nanoparticles on the lung were investigated. Upon the
alveolar epithelial cells being exposed to 12 nm silica
nanoparticles for 5 hours, the microvascular endothelium became
activated and exhibited high levels of ICAM-1 expression. It was
noted that application of 10% cyclic strain along with
nanoparticles synergistically upregulated endothelial expression of
ICAM-1. Human neutrophils circulating in the vascular channel were
seen to firmly adhere to the inflamed endothelium, to transmigrate
across the tissue barrier, and to accumulate on the epithelial
surface. These observations evidence significant proinflammatory
activities of these silica nanoparticles, which may become more
pronounced due to physiological forces that provoke acute
inflammation in the lung.
[0180] In an experiment, the present device was configured to mimic
the innate immune response to pulmonary infection of bacterial
origin. To imitate the lung afflicted with bacterial infection,
alveolar epithelial cells were apically stimulated with Escherichia
coli (E. coli) constitutively expressing green fluorescent protein
(GFP) for 5 hours. When human neutrophils were subsequently allowed
to flow in the vascular microchannel, they attached to the
endothelial cells and underwent diapedesis across the tissue
layers, indicating that bacterial stimulation of the epithelium
gave rise to endothelial activation. Upon reaching the epithelial
surface, the neutrophils showed directional movement towards
GFP-labeled bacteria and engulfed them as illustrated by detection
of phagocytosed bacteria with fluorescently labeled moving
neutrophils. It was also observed that neutrophils are capable of
ingesting more than one bacterium over short periods of time and
that their phagocytic activity continued until a majority of the
bacteria were cleared from the observation area. These results
clearly demonstrate the ability of this model to recreate the
complete process of the integrated immune response to microbial
infection within a 3D physiological organ context in vitro.
[0181] While embodiments and applications have been shown and
described, it would be apparent to those skilled in the art having
the benefit of this disclosure that many more modifications than
mentioned above are possible without departing from the inventive
concepts disclosed herein. The embodiment(s), therefore, are not to
be restricted except in the spirit of the appended claims.
[0182] The present inventive subject matter can be defined in any
of the following alphabetized paragraphs:
[0183] [A] An organomimetic device comprising:
[0184] a body having a central microchannel therein; and
[0185] an at least partially porous membrane positioned within the
central microchannel and along a plane, the membrane configured to
separate the central microchannel to form a first central
microchannel and a second central microchannel, wherein a first
fluid is applied through the first central microchannel and a
second fluid is applied through the second central microchannel,
the membrane coated with at least one attachment molecule that
supports adhesion of a plurality of living cells.
[0186] [B] The device of [A] wherein the porous membrane is at
least partially flexible, the device further comprising:
[0187] a first chamber wall of the body positioned adjacent to the
first and second central microchannels, wherein the membrane is
mounted to the first chamber wall; and
[0188] a first operating channel adjacent to the first and second
central microchannels on an opposing side of the first chamber
wall, wherein a pressure differential applied between the first
operating channel and the central microchannels causes the first
chamber wall to flex in a first desired direction to expand or
contract along the plane within the first and second central
microchannels.
[0189] [C] The device of [A] or [B] further comprising:
[0190] a second chamber wall of the body positioned adjacent to the
first and second central microchannels, wherein an opposing end of
the membrane is mounted to the second chamber wall; and
[0191] a second operating channel positioned adjacent to the
central microchannel on an opposing side of the second chamber
wall, wherein the pressure differential between to the second
operating channel and the central microchannels causes the second
chamber wall to flex in a second desired direction to expand or
contract along the plane within the first and second central micro
channels.
[0192] [D] The device of any or all of the above paragraphs wherein
at least one pore aperture in the membrane is between 0.5 and 20
microns along a width dimension.
[0193] [E] The device of any or all of the above paragraphs wherein
the membrane further comprises a first membrane and a second
membrane positioned within the central microchannel, wherein the
second membrane is oriented parallel to the first membrane to form
a third central microchannel therebetween.
[0194] [F] The device of any or all of the above paragraphs wherein
the membrane comprises PDMS,
[0195] [G] The device of any or all of the above paragraphs wherein
the membrane is coated with one or more cell layers, wherein the
one or more cell layers are applied to a surface of the
membrane.
[0196] [H] The device of any or all of the above paragraphs wherein
one or both sides of the membrane are coated with one or more cell
layers, wherein the one or more cell layers comprise cells selected
from the group consisting of metazoan, mammalian, and human
cells.
[0197] [I] The device of any or all of the above paragraphs,
wherein the cells are selected from the group consisting of
epithelial, endothelial, mesenchymal, muscle, immune, neural, and
hemapoietic cells.
[0198] [J] The device of any or all of the above paragraphs wherein
one side of the membrane is coated with epithelial cells and the
other side of the membrane is coated with endothelial cells.
[0199] [K] The device of any or all of the above paragraphs wherein
the body of the device and the membrane are made of a biocompatible
or biodegradable material.
[0200] [L] The device of any or all of the above paragraphs wherein
the device is further implanted to a living organism.
[0201] [M] The device of any or all of the above paragraphs wherein
the living organism is a human.
[0202] [N] The device of any or all of the above paragraphs wherein
the membrane is coated with the one or more cell layers in
vitro.
[0203] [O] The device of any or all of the above paragraphs,
wherein the at least one membrane is coated with the one or more
cell layers in vivo.
[0204] [P] The device of any or all of the above paragraphs,
wherein the membrane is coated with a biocompatible agent which
facilitates attachment of the at least one cell layer onto the
membrane.
[0205] [Q] The device of any or all of the above paragraphs wherein
the biocompatible agent is extracellular matrix comprising
collagen, fibronectin and/or laminin.
[0206] [R] The device of any or all of the above paragraphs wherein
the biocompatible material is selected from the group consisting of
collagen, laminin, proteoglycan, vitronectin, fibronectin,
poly-D-lysine and polysaccharide.
[0207] [S] The device of any or all of the above paragraphs wherein
the first fluid contains white blood cells.
[0208] [T] A method comprising:
[0209] selecting a organomimetic device having a body, the body
including an at least partially porous membrane positioned along a
plane within a central microchannel to partition the central
microchannel into a first central microchannel and a second central
microchannel, the membrane coated with at least one attachment
molecule that supports adhesion of a plurality of living cells;
[0210] applying a first fluid through the first central
microchannel;
[0211] applying a second fluid through the second central
microchannel; and
[0212] monitoring behavior of cells with respect to the membrane
between the first and second central micro channels.
[0213] [U] The method of any or all of the above paragraphs wherein
the membrane is at least partially elastic and the body includes at
least one operating channel positioned adjacent to the first and
second central microchannels, the method further comprising:
[0214] adjusting a pressure differential between the central
microchannels and the at least one operating channels, wherein the
membrane stretches along the plane in response to the pressure
differential.
[0215] [V] The method of any or all of the above paragraphs wherein
the adjusting of the pressure differential further comprises:
[0216] increasing the pressure differential such that one or more
sides of the membrane move in desired directions along the plane;
and
[0217] decreasing the pressure differential such that the one or
more sides of the membrane move in an opposite direction along the
plane.
[0218] [W] The method of any or all of the above paragraphs wherein
at least one pore aperture in the membrane is between 0.5 and 20
microns along a width dimension.
[0219] [X] The method of any or all of the above paragraphs further
comprising treating the membrane with one or more cell layers,
wherein the one or more cell layers are applied to a surface of the
membrane.
[0220] [Y] The method of any or all of the above paragraphs further
comprising applying one or more cell layers onto one or both sides
of the membrane, wherein the one or more cell layers comprise cells
selected from the group consisting of metazoan, mammalian, and
human cells.
[0221] [Z] The method of any or all of the above paragraphs wherein
the cells are selected from the group consisting of epithelial,
endothelial, mesenchymal, muscle, immune, neural, and hemapoietic
cells.
[0222] [AA] The method of any or all of the above paragraphs
wherein one side of the membrane is coated with epithelial cells
and the other side of the membrane is coated with endothelial
cells.
[0223] [BB] The method of any or all of the above paragraphs
wherein the body of the device and the membrane are made of a
biocompatible or biodegradable material.
[0224] [CC] The method of any or all of the above paragraphs
wherein the device is further implanted to a living organism.
[0225] [DD] The method of any or all of the above paragraphs
wherein the living organism is a human.
[0226] [EE] The method of any or all of the above paragraphs
wherein the membrane is coated with the one or more cell layers in
vitro.
[0227] [FF] The method of any or all of the above paragraphs
wherein the at least one membrane is coated with the one or more
cell layers in vivo.
[0228] [GG] The method of any or all of the above paragraphs
wherein the membrane is coated with a biocompatible agent which
facilitates attachment of the at least one cell layer onto the
membrane.
[0229] [HH] The method of any or all of the above paragraphs
wherein the biocompatible agent is extracellular matrix comprising
collagen, fibronectin and/or laminin.
[0230] [II] The method of any or all of the above paragraphs
wherein the biocompatible material is selected from the group
consisting of collagen, laminin, proteoglycan, vitronectin,
fibronectin, poly-D-lysine and polysaccharide.
[0231] [JJ] The method of any or all of the above paragraphs
wherein the first fluid contains white blood cells.
[0232] [KK] A method for determining an effect of at least one
agent in a tissue system with physiological or pathological
mechanical force, the method comprising:
[0233] selecting a device having a body, the body including an at
least partially porous membrane positioned along a plane within a
central microchannel to partition the central microchannel into a
first central microchannel and a second central microchannel;
[0234] contacting the membrane with at least one layer of cells on
a first side of the membrane and at least one layer of cells on a
second side of the porous membrane thereby forming a tissue
structure comprising at least two different types of cells;
[0235] contacting the tissue structure comprising at least two
different types of cells with the at least one agent in an
applicable cell culture medium; [0236] applying uniform or
non-uniform force on the cells for a time period; and [0237]
measuring a response of the cells in the tissue structure
comprising at least two different types of cells to determine the
effect of the at least one agent on the cells.
[0238] [LL] The method of any or all of the above paragraphs
wherein the applicable cell culture medium is supplemented with
white blood cells.
[0239] [MM] The method of any or all of the above paragraphs
wherein the uniform or non-uniform force is applied using
vacuum.
[0240] [NN] The method of any or all of the above paragraphs
wherein the tissue structure comprising at least two different
types of cells comprises alveolar epithelial cells on the first
side of the porous membrane and pulmonary microvascular cells on
the second side of the porous membrane.
[0241] [OO] The method of any or all of the above paragraphs
wherein the agent is selected from the group consisting of
nanoparticles, environmental toxins or pollutant, cigarette smoke,
chemicals or particles used in cosmetic products, drugs or drug
candidates, aerosols, naturally occurring particles including
pollen, chemical weapons, single or double-stranded nucleic acids,
viruses, bacteria and unicellular organisms.
[0242] [PP] The method of any or all of the above paragraphs
wherein the measuring the response is performed by measuring
expression of reactive oxygen species.
[0243] [QQ] The method of any or all of the above paragraphs
wherein the measuring the response is performed using tissue
staining
[0244] [RR] The method of any or all of the above paragraphs
further comprising prior to measuring the effect of the agent,
taking a biopsy of the membrane comprising tissue structure
comprising at least two different types of cells, wherein the
biopsy is stained.
[0245] [SS] The method of any or all of the above paragraphs
wherein the measuring the response is performed from a sample of
the cell culture medium in contact wherein the measuring the
response is performed from a sample of the cell culture medium in
contact with the first or the second or both sides of the membrane
form tissue structure comprising at least two different types of
cells with the first or the second or both sides of the membrane
comprising tissue structure comprising at least two different types
of cells.
[0246] [TT] The method of any or all of the above paragraphs
further comprising comparing the effect of the agent to another
agent or a control without the agent in a similar parallel device
system.
[0247] [UU] The method of any or all of the above paragraphs
further comprising a step of contacting the membrane with at least
two agents, wherein the first agent is contacted first to cause an
effect on the tissue structure comprising at least two different
types of cells and the at least second agent in contacted after a
time period to test the effect of the second agent on the tissue
structure comprising at least two different types of cells affected
with the first agent.
[0248] [VV] An organomimetic device comprising:
[0249] a body having a central microchannel; and
[0250] a plurality of membranes positioned along parallel planes in
the central microchannel, wherein at least one of the plurality of
membranes is at least partially porous, the plurality of membranes
configured to partition the central microchannel into a plurality
of central microchannels.
* * * * *