U.S. patent application number 16/125433 was filed with the patent office on 2019-01-31 for organ chips and uses thereof.
The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Anthony Bahinski, Geraldine A. Hamilton, Donald E. Ingber, Kevin Kit Parker.
Application Number | 20190032021 16/125433 |
Document ID | / |
Family ID | 48574980 |
Filed Date | 2019-01-31 |
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United States Patent
Application |
20190032021 |
Kind Code |
A1 |
Ingber; Donald E. ; et
al. |
January 31, 2019 |
Organ Chips And Uses Thereof
Abstract
Disclosed herein are organ chips that can be individually used
or integrated together to form different microphysiological
systems, e.g., for use in cell culturing, drug screening, toxicity
assays, personalized therapeutic treatment, scaffolding in tissue
repair and/or replacement, and/or pharmacokinetic or
pharmacodynamics studies.
Inventors: |
Ingber; Donald E.; (Boston,
MA) ; Parker; Kevin Kit; (Cambridge, MA) ;
Hamilton; Geraldine A.; (Boston, MA) ; Bahinski;
Anthony; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Family ID: |
48574980 |
Appl. No.: |
16/125433 |
Filed: |
September 7, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14363105 |
Jun 5, 2014 |
10087422 |
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PCT/US2012/068766 |
Dec 10, 2012 |
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16125433 |
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61569029 |
Dec 9, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 25/02 20130101;
C12N 5/0697 20130101; C12M 23/34 20130101; C12M 35/08 20130101;
C12M 23/16 20130101; C12M 35/04 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071; C12M 1/42 20060101 C12M001/42; C12M 3/06 20060101
C12M003/06; C12M 1/12 20060101 C12M001/12; C12M 1/00 20060101
C12M001/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under U01
NS073474-01 from the National Institutes of Health and Food and
Drug Administration, and W911NF-12-2-0036 from the Defense Advanced
Research Projects Agency. The government has certain rights in the
invention.
Claims
1. An in vitro microphysiological system comprising: a. at least
two different organ chips, wherein said at least two different
organ chips are selected from either one or both of the following:
(i) a first organ chip comprising: a body comprising a central
channel therein, and an at least partially porous and at least
partially flexible first membrane positioned within the central
channel and along a plane, wherein the first membrane is configured
to separate the central channel to form two sub-channels, wherein
one side of the first membrane is seeded with vascular endothelial
cells, and the other side of the first membrane is seeded with at
least one type of organ-specific parenchymal cells; (ii) a second
organ chip comprising: a body comprising a first chamber enclosing
a plurality of muscular thin films adapted to measure contraction
of muscle cells, and a second chamber comprising a layer of muscle
cells on the bottom surface of the second chamber, wherein the
bottom surface is embedded with an array of microelectrodes for
recording of action potentials, and wherein the top surface of the
second chamber is placed with at least a pair of electrodes for
providing electric field stimulation to the muscle cells; or (iii)
a combination of the first organ chip and the second organ chip;
and b. at least one connecting means between said at least two
different organ chips.
2-40. (canceled)
41. The system of claim 1, wherein the system comprises at least
three organ chips.
42. The system of claim 1, wherein the connecting means comprises a
tubing that fluidically connects an outlet of one of the organ
chips to an inlet of another organ chip.
43. The system of claim 1, wherein the first organ chip is selected
from the group consisting of a lung chip, a liver chip, a gut chip,
a kidney chip, a skin chip, a brain chip, a testis chip, and any
combinations thereof.
44. The system of claim 1, wherein the second organ chip is
selected from the group consisting of a heart chip, a skeletal
muscle chip, a lung airway smooth muscle chip, a brain chip, and
any combinations thereof.
45. The system of claim 1, wherein the first organ chip further
comprises at least a channel wall positioned adjacent to the two
sub-channels, wherein the first membrane is mounted to the channel
wall; and an operating channel adjacent to the two sub-channels on
an opposing side of the channel wall, wherein a pressure
differential applied between the operating channel and the two
sub-channels causes the channel wall to flex in a desired direction
to expand or contract along the plane within the two
sub-channels.
46. The system of claim 1, wherein the second organ chip further
comprises an at least partially porous second membrane positioned
within the first chamber to form a top chamber and a bottom
chamber, wherein the bottom chamber comprises the plurality of
muscular thin films on its bottom surface, and wherein the surface
of the second membrane in contact with the top chamber is seeded
with a layer of epithelial cells.
47. The system of claim 1, wherein the system is adapted to
determine at least one pharmacokinetic and/or pharmacodynamics
parameter of an active agent.
48. The system of claim 47, wherein the active agent is selected
from the group consisting of cells, proteins, peptides, antigens,
antibodies or portions thereof, antibody-like molecules, enzymes,
nucleic acids, siRNA, shRNA, aptamers, small molecules,
antibiotics, therapeutic agents, molecular toxins, nanomaterials,
particulates, aerosols, environmental contaminants or pollutants,
and any combinations thereof.
49. A kit comprising: a. at least one in vitro microphysiological
system of claim 1; and b. at least one agent.
50. The kit of claim 49, wherein said at least one agent comprises
a culture medium, an agent for calibration and/or validation of the
system, or a combination thereof.
51. The kit of claim 49, further comprising at least one vial of
vascular endothelial cells.
52. The kit of claim 49, further comprising at least one vial of
organ-specific parenchymal cells.
53. A method, comprising: a. providing a microfluidic device
comprising one or more microchannels comprising fluid, said
microfluidic device comprising a porous material used to construct
the device; and b. oxygenating said fluid through said porous
material used in the construction of the device.
54. The method of claim 53, wherein said microfluidic device
further comprises a membrane.
55. The method of claim 54, wherein said membrane is an at least
partially porous membrane.
56. The method of claim 53, wherein said membrane is positioned is
said one or more microchannels.
57. The method of claim 53, wherein said microfluidic device
further comprises cells within said one or more microchannels.
58. The method of claim 53, wherein said porous material comprises
PDMS.
59. A method for creating an oxygen gradient, comprising: a.
providing a microfluidic device comprising first and second
microchannels separated by a porous membrane; and b. flowing oxygen
at different concentrations through said first and second
microchannels so as to create an oxygen gradient.
60. The method of claim 59, wherein said membrane comprises
PDMS.
61. The method of claim 59, wherein said membrane comprises
cells.
62. The method of claim 59, wherein the membrane is coated with one
or more cell layers.
63. The method of claim 61, wherein said cells are liver cells.
64. The method of claim 59, further comprising monitoring oxygen
levels in at least one of said microchannels.
65. A method, comprising: a. providing a microfluidic device
comprising one or more microchannels comprising fluid; and b.
oxygenating said fluid using a gas exchange membrane.
66. The method of claim 65, wherein said one or more microchannels
comprise cells.
67. The method of claim 66, wherein said cells are contacted with
said fluid by flowing the fluid through the microchannel where the
cells are cultured.
68. The method of claim 67, wherein the fluid comprises cell
culture medium.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Application No. 61/569,029 filed Dec. 9,
2011, the content of which is incorporated herein by reference in
its entirety.
TECHNICAL FIELD OF THE DISCLOSURE
[0003] The inventions provided herein relate to organ chips and
applications thereof, e.g., analysis of drug efficacy, toxicity,
and/or pharmacodynamics using one or a plurality of the organ
chips.
BACKGROUND
[0004] Pharmacokinetics is the study of the action of
pharmaceuticals and other biologically active compounds from the
time they are introduced into the body until they are eliminated.
For example, the sequence of events for an oral drug can include
absorption through the various mucosal surfaces, distribution via
the blood stream to various tissues, biotransformation in the liver
and other tissues, action at the target site, and elimination of
drug or metabolites in urine or bile. Pharmacokinetics provides a
rational means of approaching the metabolism of a compound in a
biological system.
[0005] One of the fundamental challenges being encountered in drug,
environmental, nutritional, consumer product safety, and/or
toxicology studies includes the extrapolation of metabolic data and
risk assessment from in vitro cell culture assays to animals.
Although some conclusions can be drawn with the application of
appropriate pharmacokinetic principles, there are still substantial
limitations. One concern is that current screening assays utilize
cells under conditions that do not replicate their function in
their natural setting. The circulatory flow, interaction with other
tissues, and other parameters associated with a physiological
response are not found in standard tissue culture formats. While in
vivo animal models can be used to perform pharmacokinetics
(PK)/pharmacodynamics (PD) study, it significantly can increase the
cost of the research and the screening throughput is low.
Accordingly, there is a strong need in the art for developing
alternatives to the use of animal studies, e.g., in vitro models
that can better replicate physiological conditions for cells to
function in a similar manner as they are present in vivo. Such
models can be used, e.g., for PK/PD studies, drug screening,
engineered scaffolds for tissue/organ repair or replacement, and/or
development of a disease model of interest.
SUMMARY
[0006] One aspect provided herein relates to microengineered organ
chips or organ-on-a-chip devices. Organ chips (also known as
"organ-on-a-chip device") are microfluidic devices that are
configured to mimic at least one physiological function and/or
response of organs of interest, e.g., from a mammal (e.g., a
human), other animal or organism, an insect, or a plant. For
example, organ chips or organ-on-a-chip devices can be microfluidic
devices that comprise at least one type of living cells, e.g., at
least one type of tissue cells, cultured therein and are designed
to recapitulate the three-dimensional (3D) tissue-tissue
interfaces, mechanically active microenvironments, electrical
stimulation, chemical conditions and/or complex organ-level
functions. Examples of the organ chips described herein, can
include but are not limited to, lung chips to mimic breathing
lungs, heart chips to mimic beating hearts, liver chips to mimic
metabolic livers, kidney chips to mimic filtering kidney, gut chips
to mimic peristalsing guts, lung airway smooth muscle chips to
mimic reactive airways, skeletal muscle chips to mimic contracting
skeletal muscles, skin chips to mimic skin barriers, brain chips to
mimic blood-brain barriers, testis chips to mimic
reproductive/endocrine testes and bone marrow chips to mimic
self-renewing bone marrow.
[0007] In some embodiments, an organ chip or organ-on-a-chip device
can be configured to represent a functional microenvironment of an
organ (e.g., a functional unit or section of an organ, and/or a
tissue-capillary interface). By way of example only, a
lung-mimicking chip (or lung-on-a-chip) does not necessarily need
to mimic the structure of a whole lung. Instead, the lung-on-chip
can be configured to mimic the interaction of capillary cells and
air sac cells in an alveolus (air sac) under a mechanical
stimulation (e.g., breathing). In such embodiments, the two
different cell types (e.g., capillary cells and air sac cells) can
be cultured on opposing sides of a flexible porous membrane
disposed in a channel of a microfluidic device. The flexible porous
membrane can expand and contract to mimic the movement of an
alveolar wall during lung breathing, by controlling the pressure
gradient induced in the microfluidic device.
[0008] In some embodiments, living human cells can be cultured in
organ chips described herein to mimic at least one physiological
function and/or response of the corresponding human organs. Thus,
in one embodiment, microengineered human organ chips are also
provided herein.
[0009] A plurality of (e.g., 2 or more) organ chips representing
various organs can be assembled or connected (e.g., fluidically
connected) together to form an in vitro microphysiological system
that mimics at least one physiological function and/or response of
one or more systems in vivo, e.g., including, but not limited to, a
circulatory system, a respiratory system, an excretory system, a
nervous system, a gastrointestinal system, or any combinations
thereof. Accordingly, another aspect provided herein relates to an
in vitro microphysiological system that comprises at least two
organ chips described herein or more, e.g., at least three organ
chips, at least four organ chips or more. In some embodiments, the
in vitro microphysiological system can be used to model or study
mammalian (e.g., human) organs and physiological systems and
effects of active agents on such organs and physiological systems.
In some embodiments, the in vitro microphysiological system can be
used to model or study organs and physiological systems of other
animals (e.g., non-mammals), insects and/or plants, as well as
effects of active agents on such organs and physiological
systems.
[0010] Kits comprising a plurality of organ chips, for example, 2,
3, 4, 5, 6, 7, 8, 9, 10, or more organ chips are also provided
herein. In some embodiments, the organ chips in the kit can be all
the same, i.e., corresponding to the same organ. In some
embodiments, at least some of the organ chips in the kit can
represent a different organ. For example, a kit directed to a
circulatory system can comprise at least one heart chip and at
least one bone-marrow chip. In an alternative embodiment, a kit
directed to a gastrointestinal system can comprise at least one
liver chip and at least one gut chip. Depending on the
microphysiological system of interest, the kits can comprise a
plurality of distinct organ chips that are involved in the
microphysiological system.
[0011] In some embodiments, the organ chips can each be
individually packaged, e.g., for sterility purposes. In some
embodiments, the kits can further comprise at least one agent,
e.g., an appropriate culture medium for each different organ chip.
In some embodiments, the kits can further comprise an instruction
manual, e.g., instructions on connecting various organ chips
together to form an integrated network.
[0012] The organ chips, microphysiological systems and/or kits
described herein can be used for various applications where
simulation of a physiological condition is desirable, e.g., drug
screening, PK/PD studies, engineered scaffolds for tissue/organ
repair or replacement, and/or development of a disease model of
interest. In some embodiments, the cells cultured in the organ
chips can be collected from a subject, e.g., for personalized
therapeutic treatment. For example, subject-specific cells can be
cultured in an organ chip or a microphysiological system simulated
for a disease or disorder that the subject is diagnosed of, or
suspected of having, and subjected to various kinds and/or dosages
of drugs to determine an optimal treatment regimen for the
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is an image showing one embodiment of a
lung-on-a-chip described herein, inside which the breathing of a
lung is stimulated. In this embodiment, the lung-on-a-chip
comprises ports for nutrient delivery, waste disposal, and/or
creation of a pressure gradient to mimic breathing.
[0014] FIGS. 2A-2B are schematic representations of a lung-on-a
chip in accordance with one embodiment described herein. FIG. 2A is
a schematic representation showing an exemplary configuration of a
lung-on-a-chip. FIG. 2B is a schematic representation showing
movement (e.g., stretching) of a flexible porous membrane under
application of vacuum to side chambers of the lung-on-a-chip.
[0015] FIGS. 3A-3C shows that human lung-on-a-chip can be used to
predict TL-2 chemotherapy toxicity (vascular leakage) responses
based on mimicry of the lung's dynamic mechanically-active
(breathing) microenvironment.
[0016] FIGS. 4A-4D is a set of images showing that a contractile
heart (muscle) chip mimics tissue organization (FIG. 4A) in a
multiplexed array of "muscular thin films" (MTFs) within a
microfluidic device (FIGS. 4B and 4C), which can be used to
quantitate contractile stress in real-time (FIG. 4D).
[0017] FIG. 5 is a set of line graphs showing that "muscular thin
films" MTFs mimic whole heart tissue drug responses. Top row shows
the dose response of engineered neonatal rat ventricular tissues in
the form of muscular thin films on the heart chip, treated with
calcium (left), caffeine (middle), and isoproterenol (right). The
bottom row shows the corresponding responses of adult rat
ventricular strips.
[0018] FIG. 6 is a schematic representation showing a series of
exemplary assembly steps for one embodiment of a muscular thin film
contractility assay based on a PDMS thin film.
[0019] FIG. 7 is a schematic representation showing a series of
exemplary assembly steps for one embodiment of a muscular thin film
contractility assay based on a patterned alginate thin film.
[0020] FIG. 8 is a schematic diagram showing exemplary features of
a heart chip according to one embodiment described herein. (i)
depicts a dual-chamber system with a single medium stream that
feeds 2 chambers: an electrophysiological (EPhys) chamber and a MTF
chamber; (ii) shows that the EPhys chamber can allow
electrophysiological recordings on a monolayer of cardiac muscle in
a low volume chamber with micro-electrodes embedded in the bottom
of the chamber; (iii) illustrates that a larger chamber situated
next to the EPhys chamber can allow high throughput contractility
measurements using an array of muscular thin films; (iv) shows that
the MTF chamber consists of an anisotropic layer of cardiac
myocytes cultured on laser-cut horizontal MTFs whose radius of
curvature can be measured optically.
[0021] FIGS. 9A-9B is a set of schematic diagrams showing an
alternative technology to monitor cellular contraction. FIG. 9A
shows that cell monolayers are adhered to a deformable, perforated
membrane within a microfluidic device (i & ii). FIG. 9B shows
that as the muscle cell layer contracts, the substrate deforms,
such that the morphology of the holes within the substrate is
altered (i & ii). Hence, the morphology of the holes within the
substrate can be monitored optically to determine the state of
cellular contraction, with undeformed holes representing the
relaxed state (FIG. 9A, ii) and deformed holes representing the
contracted state (FIG. 9B, ii). The eccentricity of the holes can
be evaluated with specifically-programmed algorithms, e.g.,
software (e.g., DBG software) originally designed to quantify
nuclear eccentricity.
[0022] FIGS. 10A-10D is a set of data showing physiological
responses of human vascular smooth muscle (VSM) and engineered rat
cardiac muscle (CM) on the same chip to drugs determined by a MTF
assay. FIG. 10A is a schematic representation showing manufacture
of an organ chip with two different kinds of muscle (striated and
smooth). FIG. 10B is a set of data showing MTF deformation (i, v,
ix), CM diastole (ii, vi, x), CM peak systole (iii, vii, xi), and
CM stress (iv, viii, xii), before and after exposure to drugs
(e.g., ET-1, and ROCK inhibitor) during the contractility assay.
FIGS. 10C and 10D are data graphs showing the contractility of the
VSM is considerably slower than the CM and the stress histories are
depicted uniquely for each one.
[0023] FIGS. 11A-11F are images showing a human gut chip according
to one embodiment described herein. FIG. 11A is a schematic
representation showing a portion of a human gut chip that mimics
normal villus architecture of the intestine. FIGS. 11B-11D shows
that a gut chip can mimic normal villus architecture of the
intestine, in part by leveraging the lung-on-a-chip (or lung chip)
mechanically activated, multi-layered microfluidic architecture
(FIG. 11B) to rhythmically distort the epithelium as normally
occurs during peristalsis (FIGS. 11C-11D). FIG. 11E is an image
showing perfusion of cells in the device. FIG. 11F is an image
showing perfusion of cells in the device can maintain cell
viability for weeks and result in formation of villi that take the
height of the Interstitial Channel.
[0024] FIG. 12 is a schematic diagram showing architecture of human
blood-brain-barrier and an organ chip that mimics the
blood-brain-barrier. In one embodiment, the normal architecture of
the human blood brain barrier (top panel) is mimicked by culturing
human endothelium on one side of a porous membrane and human
astrocytes on the other side within a microfluidic channel with
platinum electrodes embedded therein (bottom panel).
[0025] FIG. 13 is a schematic diagram showing exemplary features of
a skeletal muscle chip according to one embodiment described
herein. (i) depicts a dual-chamber system with a single medium
stream that feeds 2 chambers: an electrophysiological (EPhys)
chamber and an MTF chamber; (ii) shows that the EPhys chamber can
allow EMG recordings on a monolayer of skeletal muscle in a low
volume chamber with micro-electrodes embedded in the bottom of the
chamber; (iii) shows that a larger chamber situated next to the
EPhys chamber can allow high throughput contractility measurements
using an array of muscular thin films (MTFs); (iv) shows that the
MTF chamber consists of an anisotropic layer of skeletal myocytes
cultured on laser-cut horizontal MTFs whose radius of curvature can
be measured optically.
[0026] FIG. 14 is a schematic representation of functional readouts
from a co-culture of skeletal muscle and adipocyte layers, for
example, using the skeletal muscle chip shown in FIG. 13. (i) shows
an electrophysiological (EPhys) chamber, which allows EMG
recordings on a monolayer of a co-culture of adipose and skeletal
muscle. (ii) shows a MTF chamber with an array of muscular thin
films (MTFs) built from a heterogeneous co-culture of skeletal
muscle and adipose tissue.
[0027] FIGS. 15A-15C are data showing anisotropic cardiac tissue
formation on micromolded alginate substrates. FIG. 15A is data
showing that the micromolding technique replicates faithfully the
original pattern. FIG. 15B is a phase contrast image of
representative tissues. FIG. 15C is an immunofluorescence composite
image of muscular thin films: actin is red, nuclei are blue and
.alpha.-actinin is green. Scale bar equals 50 .mu.m. In some
embodiments, alginate micromolded surfaces can be used to align and
culture skeletal muscle cells (e.g., myotubes) in a 3-D like
environment, instead of a 2-D flat substrate.
[0028] FIGS. 16A-16B is a set of schematic representation showing
an airway chip according to one embodiment described herein. FIG.
16A is a schematic diagram showing an airway on a chip, in one
embodiment, can comprise healthy bronchial tissue, cultured in
liquid media (i), e.g., with a capability of drug perfusion (ii).
The cell monolayer can exhibit a linear arrangement of cells,
adhered to the top surface of a PDMS muscular thin film. Incubation
in culture media (no drug or test agent) can yield relaxed
bronchial thin films (iii), while incubation with drugs can yield
contracted bronchial thin films (iv). The contractility can be
measured for grading the drug response in the tissue. FIG. 16B is a
schematic representation of exemplary dimensions of an airway chip
containing multiple bronchial thin films (i), with an optional
capability to add a layer of epithelial columnar cells (ii). This
cell layer can be adhered to a porous membrane that separates the
bronchial from epithelial cells, and be exposed to air flow,
aerosols or a combination thereof.
[0029] FIGS. 17A-17B are schematic representation showing exemplary
features of an airway chip with two chambers in accordance with one
embodiment described herein. FIG. 17A is a schematic representation
of an airway on a chip with two chambers. Chamber 1 can contain
healthy bronchial tissue, while an asthmatic phenotype can be
contained in Chamber 2. The asthmatic phenotype can comprise
cultures from human diseased cells or cells that are induced
artificially (e.g., by toxic agents, temperature) to display at
least one phenotype of diseased cells Chambers 1 and 2 can be
cultured in liquid media (i) with an optional capability of drug
perfusion (ii). Media and/or drugs can be kept separate between
Chamber 1 and Chamber 2 by the closing of a valve (pictured in the
legend, with a single pole, single throw (SPST) and Normal
Open/Normal Close (NO/NC) valve). Monolayers of healthy and
asthmatic cells can exhibit an anisotropic organization and can be
adhered to the top surface of PDMS muscular thin films. Incubation
in culture media can yield relaxed thin films (iii), while
incubation with drugs can yield contracted thin films (iv). The
contractility can be measured for grading the response of the
different tissue types to the drugs. FIG. 17B is a schematic
representation of exemplary dimensions of an airway chip containing
multiple bronchial thin films (i), with an optional capability to
add a layer of epithelial columnar cells (ii). This epithelial cell
layer can be adhered to a porous membrane that separates the
bronchial muscle from the epithelium, and be exposed to air flow,
aerosols, or a combination thereof.
[0030] FIGS. 18A-18B are schematic diagrams showing an alternative
technology to monitor cellular contraction. FIG. 18A shows that
cell monolayers can be adhered to a "swiss cheese"-like substrate
or a deformable, perforated membrane, situated within a
microfluidic device (i & ii). FIG. 18B shows that as the muscle
cell layer contracts, the substrate deforms, such that the
morphology of the holes within the substrate is altered (i &
ii). The morphology of the holes within the substrate can be
monitored optically to determine the state of the cellular
contraction, with undeformed holes representing the relaxed state
(FIG. 18A, ii) and deformed holes representing the contracted state
(FIG. 18B, ii). The eccentricity of the holes can be evaluated with
a specifically-programmed algorithm, e.g., software (e.g., DBG
software) originally designed to quantify nuclear eccentricity.
[0031] FIGS. 19A-19B shows an exemplary setup of a multiple film
chip experiment and collected data from a human asthma (e.g., a
chemically-induced human asthma) induced on one embodiment of an
organ chip. FIG. 19A is a schematic diagram showing exemplary
manufacture of an organ chip comprising human bronchial smooth
muscle thin films. FIG. 19B show data comparing the phenotypes
and/or behavior of control cells and diseased cells (induced with
asthma) in the organ chip. (i) and (ii) show actin staining of
healthy engineered tissue and the chemically induced asthma model,
respectively. Differences in actin alignment within the tissue
constructs, as indicated by the orientational order parameter (iii)
indicates significant remodeling of the contractile apparatus. Drug
experiments (iv for healthy and v for asthma model) indicate
differences in the contractile response to acetylcholine (AcH) and
to a Rho kinase inhibitor (HA 1077). (vi) is a plot graph showing
percentages of cell contraction and relaxation for control
(healthy) and asthma models.
[0032] FIG. 20 is a set of data showing that a synthetic bone
marrow (sBM) can fully recapitulate natural mouse bone marrow (mBM)
but not peripheral blood (mPB) 8 weeks after implanting an organ
chip with differentiated blood cells (DMPs) or hematopoietic stem
and progenitor stem cells (BMPs) subcutaneously. Similar
functionality can be maintained in vitro by culturing in a
microfluidic device the sBM explant.
DETAILED DESCRIPTION OF THE INVENTION
[0033] There is a need in developing alternative models to in vivo
animal models for various applications, e.g., in analysis of drug
efficacy, toxicity, and/or pharmacodynamics, or in studies of
diseases or disorders. To this end, the inventors have developed
various designs and configurations of "organ chips" (also used
interchangeably herein with the term "organ-on-a-chip devices"),
which can be configured as microfluidic devices to mimic at least
one physiological function and/or response of different organs, and
can be used to create in vitro microphysiological systems. For
example, organ chips or organ-on-a-chip devices can be microfluidic
devices that comprise at least one type of living cells (e.g.,
mammalian cells such as human cells) cultured therein and are
designed to recapitulate the three-dimensional (3D) tissue-tissue
interfaces, mechanically active microenvironments, electrical
stimulation, chemical conditions and/or complex organ-level
functions. Examples of the organ chips described herein, include,
but are not limited to, lung chips to mimic breathing lung, heart
chips to mimic beating heart, liver chips to mimic metabolic liver,
kidney chips to mimic filtering kidney, gut chips to mimic
peristalsing gut, lung airway smooth muscle chips to mimic reactive
airway, skeletal muscle chips to mimic contracting skeletal muscle,
skin chips to mimic skin barrier, brain chips to mimic blood-brain
barrier, testis chips to mimic reproductive/endocrine testis, bone
marrow chips to mimic self-renewing bone marrow, and any
combinations thereof.
[0034] In some embodiments, an organ chip or organ-on-a-chip device
described herein can be configured to represent a functional
microenvironment of an organ (e.g., a functional unit or section of
an organ, and/or a tissue-capillary interface), e.g., but not
limited to, an alveolar-capillary interface of a lung, a
blood-brain-barrier of a brain, or a skin bather of a skin. By way
of example only, a lung-mimicking chip (or lung-on-a-chip), which
is further described below, does not necessarily need to mimic the
structure of a whole lung. Instead, the lung-on-chip can be
configured to mimic the interaction of capillary cells and air sac
cells in an alveolus (air sac) under a mechanical stimulation
(e.g., breathing). In such embodiments, the two different cell
types (e.g., capillary cells and air sac cells) can be cultured on
opposing sides of a flexible porous membrane disposed in a channel
of a microfluidic device. The flexible porous membrane can expand
and contract to mimic the movement of an alveolar wall during lung
breathing, by controlling the pressure gradient induced in the
microfluidic device.
[0035] The organ chips can be used, individually or connected
together (e.g., fluidically connected), for various applications
where simulation of a physiological condition is desirable, e.g.,
drug screening, pharmacokinetics (PK)/pharmacodynamics (PD)
studies, engineered scaffolds for tissue/organ repair or
replacement, development of a disease model, and/or personalized
therapeutic treatment.
[0036] As used herein, the term "fluidically connected" refers to
two or more organ chips connected in an appropriate manner such
that a fluid or a least a portion of a fluid (e.g., any flowable
material or medium, e.g., but not limited to, liquid, gas,
suspension, aerosols, cell culture medium, and/or biological fluid)
can directly or indirectly pass or flow from one organ chip to
another organ chip. In some embodiments, two or more organ chips
can be fluidically connected together, for example, using one or
more fluid-transfer connecting means (e.g., adaptors, tubing,
splitters, valves, pumps and/or channels) between the two or more
organ chips. For example, two or more organ chips can be
fluidically connected by connecting an outlet of one organ chip to
an inlet of another organ chip using tubing, a conduit, a channel,
piping or any combinations thereof. In some embodiments, two or
more organ chips can be fluidically connected by, e.g., at least
one pumping device and/or at least one valve device. In some
embodiments, the pumping device and/or valve device can be
configured for microfluidic applications, e.g., the membrane-based
fluid-flow control devices as described in U.S. Provisional
Application No. 61/735,206 filed Dec. 10, 2012, the content of
which is incorporated herein by reference in its entirety.
[0037] In other embodiments, two or more organ chips can be
fluidically connected together when one or more other connecting
means (e.g., devices, systems, and/or modules that can perform an
additional function other than fluid transfer, e.g., but not
limited to, filtration, signal detection, and/or imaging) are
present between the two or more organ chips. In these embodiments,
by way of example only, two or more organ chips can be fluidically
connected, when the two or more organ chips are indirectly
connected, e.g., through a biosensor, a filter, and/or an
analytical instrument (e.g., via tubing), such that a fluid exiting
the previous organ chip can be detoured to first flow through the
biosensor, filter and/or analytical instrument, e.g., for
detection, analysis and/or filtration of the fluid, before it
enters the next organ chip. In these embodiments, at least a
portion of the fluid can pass or flow from one organ chip to
another organ chip. In some embodiments, two or more organ chips
can be fluidically connected by, e.g., at least one bubble trap,
e.g., the bubble trap can be a membrane-based bubble trap as
described in U.S. Provisional Application No. 61/696,997, filed
Sep. 5, 2012, and U.S. Provisional Application No. 61/735,215
titled "Cartridge Manifold and Membrane Based-Microfluidic Bubble
Trap," filed on Dec. 10, 2012, the contents of both of which are
incorporated herein by reference in their entireties.
Alternatively, two or more organ chips can be connected such that a
fluid can pass or flow directly from one organ chip to another
organ chip without any intervening components. In such an
embodiment, the two or more organ chips can be designed and/or
integrated on the same chip such that the outlet of one organ chip
and the inlet of another organ chip share the same port.
[0038] In some embodiments, one or more organ chips (e.g., heart
chips) described herein can be adapted to fluidically connected
upstream and/or downstream to at least one or more different organ
chips (e.g., but not limited to lung chips or liver chips) to form
an in vitro microphysiological system, which can be used to
determine biological effects (e.g., but not limited to, toxicity,
and/or immune response) of active agents on more than one organs.
Examples of active agents include, but are not limited to, cells
(including, e.g., but not limited to, bacteria and/or virus),
proteins, peptides, antigens, antibodies or portions thereof,
enzymes, nucleic acids, siRNA, shRNA, aptamers, small molecules,
antibiotics, therapeutic agents, molecular toxins, nanomaterials,
particulates, aerosols, environmental contaminants or pollutants
(e.g., but not limited to, microorganisms, organic/inorganic
contaminants present in food and/or water, and/or air pollutants),
and any combinations thereof. In some embodiments, the in vitro
microphysiological system can be used to evaluate active agents
that are effective in treating a disease or disorder in an organ,
but might be toxic to other organ systems. For example, a drug,
e.g., Ventolin, known to treat or prevent bronchospasm in subjects
with reversible obstructive airway disease can be toxic to or
adversely affect heart function. Thus, integration of two or more
organ chips to form an in vitro microphysiological system can allow
for testing or screening of drugs that are effective in treatment
of a certain disease or disorder with minimal side effects or
undesirable effects on other organs.
[0039] Accordingly, in another aspect, provided herein are
integrated network or functional in vitro microphysiological
systems, each of which mimics at least one physiological function
and/or response of one or more systems in vivo, e.g., of a mammal
(e.g., a human), other animal, insect and/or plant. In some
embodiments, the in vitro microphysiological systems described
herein can mimic at least one physiological function and/or
response of one or more systems in vivo, e.g., of a mammal (e.g., a
human), including, e.g., but not limited to, a circulatory system,
a respiratory system, an excretory system, a nervous system, a
gastrointestinal system, or any combinations thereof. The in vitro
microphysiological systems described herein are generally formed by
connecting (e.g., fluidically connecting) together at least two
organ chips representing different organs described herein.
Different combinations of organ chips can be used in the system for
different applications. In some embodiments, a plurality of organ
chips (e.g., at least 1, at least 2, at least 3, at least 4, at
least 5 or more organ chips) can be fluidically connected, e.g.,
via a tubing, to each other to form a microphysiological system,
e.g., a circulatory system (comprising a heart chip with vascular
endothelium and a bone marrow chip), a respiratory system
(comprising a lung chip, and an airway smooth muscle chip), an
immune system (comprising a bone marrow chip with other immune
cells, e.g., macrophages); a musculoskeletal system (comprising a
skeletal muscle chip), an excretory system (comprising a lung chip,
a gut chip, and a kidney chip), an urinary system (comprising a
bladder chip and a kidney chip), a nervous system (comprising a
brain chip with astrocytes and neuronal networks), a reproductive
system (comprising testis chip), an endocrine system (comprising a
testis chip), a gastrointestinal system (comprising a liver chip,
and a gut chip), an integumentary system (comprising a skin chip),
and a urinary system (comprising a kidney chip).
[0040] Depending on various target applications, e.g., for use as a
disease model or for pharmacokinetics study of a drug, different
combinations of organ chips can be selected. For example, in one
embodiment, Lung Chips, Heart Chips and Liver Chips can be selected
to form an in vitro microphysiological system, e.g., for
determination of clinically relevant pharmacokinetics
(PK)/pharmacodynamics (PD) as well as efficacy and toxicity (e.g.,
cardiotoxicity, which is the cause of more than 30% of all drug
failures).
[0041] In some embodiments, the in vitro microphysiological system
can further comprise a bone marrow chip fluidically connected to
the at least two different organ chips. In one embodiment, the bone
marrow chip described in the International Appl. No.
PCT/US12/40188, the content of which is incorporated herein by
reference in its entirety, can be utilized in the in vitro
microphysiological system described herein.
[0042] In some embodiments, the in vitro microphysiological system
can further comprise a spleen chip fluidically connected to the at
least two different organ chips. In one embodiment, the spleen chip
described in the International Appl. No. WO 2012/135834, the
content of which is incorporated here by reference in its entirety,
can be utilized in the in vitro microphysiological system described
herein.
[0043] In some embodiments, the in vitro microphysiological systems
comprising a combination (e.g., at least 2 or more) of different
organ chips can be disposed in a housing and/or the universal
cartridges that can hold one or more organ chips as described in
the U.S. Provisional Appl. Nos. 61/569,004 filed Dec. 9, 2011 and
61/696,997 filed Sep. 5, 2012, the contents of which are
incorporated herein by reference in their entireties. For example,
a housing to enclose various combinations of organ chips therein
can provide functionalities, e.g., but not limited to temperature
control, nutrient replenishment, pressure adjustment, imaging,
sample analysis, and/or any combinations thereof.
[0044] An organ chip can also include a microfluidic device which
can mimic at least one physiological function of at least one
living organ from a mammal (e.g., human), other animal, insect or
plant. In some embodiments, an organ chip can be a microfluidic
device which can mimic at least one physiological function of one
mammalian (e.g., human) organ. In some embodiments, an organ chip
can be a microfluidic device which can mimic physiological function
of at least one (including 1, 2, 3, 4, 5, 6, 7 or more) mammalian
(e.g., human) organs. In some embodiments where the organ chips
mimic physiological functions of more than one mammalian (e.g.,
human) organs, the organ chips can comprise individual sub-units,
each of which can mimic physiological function of one specific
mammalian (e.g., human) organ.
[0045] In some embodiments, the in vitro microphysiological system
can comprise at least two different organ chips (e.g., each organ
chip representing a different organ) and at least one or more
connecting means (e.g., at least two or more connecting means)
between the at least two different organ chips. The at least two
different organ chips can be selected from one or both of the
following design and/or configuration: (i) a first organ chip can
comprise: a body comprising a central channel therein, and an least
partially porous and at least partially flexible first membrane
positioned within the central channel and along a plane, wherein
the first membrane is configured to separate the central channel to
form two sub-channels, wherein one side of the first membrane is
seeded with vascular endothelial cells, and the other side of the
first membrane is seeded with at least one type of organ-specific
parenchymal cells; and (ii) a second organ chip can comprise: a
body comprising a first chamber enclosing a plurality of muscular
thin films adapted to measure contraction of muscle cells, and a
second chamber comprising a layer of muscle cells on the bottom
surface of the second chamber, wherein the bottom surface is
embedded with an array of microelectrodes for recording of action
potentials, and wherein the top surface of the second chamber is
placed with at least a pair of electrodes for providing electric
field stimulation to the muscle cells.
[0046] In some embodiments, the at least two different organ chips
can comprise at least two or more (e.g., 2, 3, 4, 5, or more) said
first organ chips described herein. In one embodiment, the at least
to different organ chips can comprise a lung chip described herein
and a gut chip described herein. In one embodiment, the at least to
different organ chips can comprise a lung chip described herein and
a liver chip described herein.
[0047] In some embodiments, the at least two different organ chips
can comprise at least two or more (e.g., 2, 3, 4, 5, or more) said
second organ chips described herein.
[0048] In some embodiments, the at least two different organ chips
can comprise at least one (e.g., 1, 2, 3, 4, 5 or more) said first
organ chips described herein and at least one (e.g., 1, 2, 3, 4, 5,
or more) said second organ chips described herein.
[0049] The design and/or configuration of the first and second
organ chips described herein generally provide the basis for
development and construction of various organ chips, e.g., but not
limited to, lung chips, liver chips, gut chips, kidney chips, heart
chips, skin chips, brain chips, testis chips, skeletal muscle
chips, lung airway smooth muscle chips ("airway chips"), and any
combinations thereof. The application and modifications of these
two basic organ chip designs to create various organ chips are
illustrated and described in the section below "Examples of organ
chips or organ-on-a-chip devices." As described below, in some
embodiments, the organ chips can be designed to have a common shape
and have positioned inlets and outlets for delivery of fluids to
the Microvascular and Interstitial fluid channels lined by
microvascular endothelium and organ-specific parenchymal cells
(e.g., but not limited to, alveolar epithelium, heart muscle,
hepatocytes), respectively.
[0050] Organ chips generally comprise a base substrate and at least
one channel disposed therein. The number and dimension of channels
in an organ chip can vary depending on the design, dimension and/or
function of the organ chip. In some embodiments, an organ chip can
comprise a plurality of channels (e.g., at least two, at least
three, at least four, at least five, at least six, at least seven,
at least eight, at least nine, at least ten or more channels). One
of skill in the art will readily be able to design and determine
optimum number and/or dimension of channels required to achieve a
certain application. For example, if assessment of reproducibility
and/or comparison of at least two experimental conditions are
desirable, an organ chip can be constructed to comprise at least
two, at least three, at least four, at least five identical
channels. This can provide for a number of read-outs per chip,
e.g., allowing assessment of reproducibility and/or for validation
and implementation of the technology. For example, each channel can
run a different condition (e.g., culturing normal (healthy) cells
vs. diseased cells in different channels, or applying different
dosages of the same drug to different channels, or applying
different drugs at the same dosage to different channels). In some
embodiments, an organ chip can comprise at least two parallel
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) channels. In one
embodiment, an organ chip comprises four parallel channels, e.g.,
four identical parallel channels. Without wishing to be bound by
theory, this configuration can provide quadruplicate read-outs per
chip.
[0051] The dimensions of the channels in the organ chips can each
independently vary, e.g., depending on the channel function (e.g.,
as a conduit for fluid transfer or as a chamber for cell culture,
e.g., for subsequent monitoring of cellular response), flow
conditions, tissue microenvironment to be simulated, and/or methods
for detecting cellular response. Thus, the cross-sectional
dimensions of the channels can vary from about 10 .mu.m to about 1
cm, or from about 100 .mu.m to about 0.5 cm.
[0052] In some embodiments, at least a portion of the channels
disposed in the organ chips can comprise cells. In these
embodiments, the channels can each be independently lined by one
layer or multilayers of organ-specific parenchymal cell types (or
differentiated cells) and/or vascular endothelium (a layer of
vascular endothelial cells) in relevant tissue microenvironment
(e.g., mechanochemical microenvironments), with or without
intervening connective tissue cells (e.g., fibroblasts, smooth
muscle cells, mast cells) or immune cells (e.g., neutrophils,
macrophages).
[0053] The organ chips can be sized to a specific need, e.g., for
high throughput drug screening, or scaffolding, e.g., for tissue
repair and/or replacement. In some embodiments, the organ chips can
be implantable, and thus they can be sized to suit a target
implantation site.
[0054] In some embodiments, the organ chips can be fabricated from
any biocompatible materials. Examples of biocompatible materials
include, but are not limited to, glass, silicons, polyurethanes or
derivatives thereof, rubber, molded plastic, polymethylmethacrylate
(PMMA), polycarbonate, polytetrafluoroethylene (TEFLON.TM.),
polyvinylchloride (PVC), polydimethylsiloxane (PDMS), and
polysulfone. In one embodiment, organ chips can be fabricated from
PDMS (poly-dimethylsiloxane). In some embodiments, the organ chips
can be disposable. In some embodiments, the organ chips can be
fabricated from one or more materials that allow sterilization
(e.g., by UV, high temperature and/or pressure, ethylene oxide, or
ethanol) after use.
[0055] In some embodiments, at least one channel of the organ chips
can comprise one or more membranes, e.g., at least 1, at least 2,
at least 3 or more membranes to separate the channel into
sub-channels. The membrane can be rigid or at least partially
flexible. The term "flexible" as used herein refers to a membrane
that can be stretched and/or contracted by at least about 3%, at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 50%, at least about
60% or more, of its original length, without causing any
macroscopic breaking, when a pressure is applied. In some
embodiments, a flexible membrane can fully or partially restore to
its original length after the pressure is released.
[0056] In some embodiments, the membrane can be non-porous or at
least partially porous. In some embodiments, the pore size of the
membrane can be large enough to allow cells pass through it. In
some embodiments, the pore size of the membrane can be too small
for cells to pass through, but large enough for nutrient or fluid
molecules to pass through or permeate.
[0057] In some embodiments, the membrane can be non-coated or
coated with extracellular matrix molecules (ECM) to facilitate cell
adhesion (e.g., but not limited to, fibronectin, collagen,
Matrigel, laminin, vitronectin, and/or any combinations thereof),
other proteins such as growth factors or ligands (e.g., to
facilitate cell growth and/or cell signaling). In some embodiments,
the surface of the membrane can be modified and/or activated, e.g.,
with any art-recognized polymer surface modification techniques
such that bioactive molecules, e.g., ECM molecules, carbohydrates,
proteins such as growth factors or ligands, can be covalently or
non-covalently attached to or coated on it. Examples of polymer
surface modification such as wet chemical, organosilanization,
ionized gas treatments, and UV irradiation can be used to modify
the membrane to permit covalent conjugation of bioactive molecules
to the modified surfaces, such as usage of hydrophilic,
bifunctional, and/or branched spacer molecules. See, e.g., Goddard
and Hotchkiss "Polymer surface modification for the attachment of
bioactive compounds" Progress in Polymer Science, Volume 32, Issue
7, July 2007, Pages 698-725, for examples of polymer surface
modification techniques.
[0058] The material for the membrane can be selected for at least
one of the following properties, but are not limited to: the
material is (i) biocompatible, (ii) complies with IS 10993-5 (in
vitro cytotoxicity tests for medical devices), (iii) has low
absorption of hydrophobic dye/drug and other chemical compounds,
(iv) is cell adhesive, (v) is optically clear, is highly flexible,
moldable, bondable, (vi) has low autofluorescence, (vii) does not
swell in water, or (viii) has any combinations of the
aforementioned properties. In one embodiment, the membrane material
can include polyurethane (e.g., Clear flex 50 polyurethane). In
another embodiment, the membrane material can include PDMS.
[0059] In some embodiments, the membrane can be seeded with or
without cells. In some embodiments where cells are seeded on the
membrane, cells can be seeded on one side or both sides of the
membrane. In some embodiments, both sides of the membrane can be
seeded with the same cells. In other embodiments, both sides of the
membrane can be seeded with different cells, as described below,
e.g., to create a Microvascular channel (comprising vascular
endothelial cells) and an Interstitial channel (comprising
organ-specific parenchymal cells). In some embodiments, the
membrane can be seeded with at least one layer of cells, including,
at least 2 layers of cells or more. Each layer of cells can be the
same or different.
[0060] In some embodiments, at least one channel or sub-channel of
the organ chip can be filled with a gel or a hydrogel, e.g., but
not limited to, collagen gel, matrigel gel, fibrin gel, or any
combinations thereof. The gel can be seeded with or without
cells.
[0061] In some embodiments, at least one channel or sub-channel of
the organ chip can contain a tissue, e.g., a biopsy collected from
a subject.
[0062] In some embodiments, the inner surface(s) of the channel(s)
(or channel walls) and/or membrane(s) that are in contact with a
fluid (e.g. a liquid or a gas) can be modified for reducing
non-specific binding of a species in the fluid to the inner
surface(s) of the channel(s). For example, at least one surface of
the channel(s) and/or membrane(s) in contact with the fluid can be
coated with a surfactant, e.g., PLURONIC.RTM. 127, or a blocking
protein such as bovine serum albumin, for reducing cell or protein
adhesion thereto. Additional surfactant that can be used to reduce
the adhesive force between the surface of the channel and
non-specific binding of a species in a fluid sample can include,
but are not limited to, hydrophilic (especially amphipathic)
polymers and polymeric surface-acting agents; non-ionic agents such
as polyhydric alcohol-type surfactants, e.g., fatty acid esters of
glycerol, pentaerythritol, sorbitol, sorbitan, and more hydrophilic
agents made by their alkoxylation, including polysorbates
(TWEEN.RTM.); polyethylene glycol-type surfactants such as PLURONIC
surfactants (e.g., poloxamers), polyethylene glycol (PEG),
methoxypolyethylene glycol (MPEG), polyacrylic acid,
polyglycosides, soluble polysaccharides, dextrins, microdextrins,
gums, and agar; ionic agents, including anionic surfactants such as
salts of carboxylic acids (soaps), sulfuric acids, sulfuric esters
of higher alcohols; cationic surfactants such as salts of
alkylamine type, quaternary ammonium salts, or amphoteric
surfactants such as amino acid type surfactants and betaine type
surfactants. A skilled artisan will readily be able to determine
appropriate methods and/or reagents for use to reduce non-specific
binding of a species in a fluid to the channel wall(s) and/or
membranes, based on the substrate material of the microfluidic
devices and/or types of species to be blocked.
[0063] In some embodiments, there can be at least one micro-post,
including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more micro-posts within
one or more channels. The dimension and/or arrangement of the
micro-posts can be determined by a user. For example, the
micro-posts can be used to separate cell debris from a flowing
fluid to prevent clogging the downstream channel. In some
embodiments, the micro-posts can be coated with an agent (e.g.,
antibodies) that permits capture of specific cells.
[0064] In some embodiments, the organ chips can comprise a
plurality of ports. For example, the organ chips can comprise at
least one inlet port for introducing culture medium, nutrients or
test agents such as drugs into the organ chips, and at least one
outlet port for a fluid to exit. In some embodiments, at least one
port can be connected to a pump or a syringe, e.g., via a tubing,
to facilitate the fluid transfer through the channel and/or to
apply a pressure to the channel. In some embodiments, at least one
port can be connected to at least one electrical component, e.g.,
an electrode for ECG measurement. In some embodiments, at least one
port can be connected to a nebulizer, e.g., to generate aerosolized
liquid for aerosol delivery. In some embodiments, at least one port
can be connected to or interfaced with a processor, which stores
and/or analyzes the signal from a biosensor incorporated therein.
The processor can transfer the data to computer memory (either hard
disk or RAM) from where it can be used by a software program to
further analyze, print and/or display the results. In some
embodiments, the organ chips can have control ports, e.g., for
application of mechanical deformation (e.g., side chambers to apply
cyclic vacuum, as in the Lung Chip described herein and in the
International Application No.: WO 2010/009307, the contents of
which are incorporated herein by reference in their entireties)
and/or electrical connections (e.g., for electrophysiological
analysis of muscle and nerve conduction).
[0065] In some embodiments of any types of organ chips and/or in
vitro microphysiological systems described herein, any fluid
control elements can be incorporated into the organ chips and/or in
vitro microphysiological systems to modulate the fluid flow. For
example, bubble traps can be integrated into each organ chip and/or
in vitro microphysiological systems to minimize the effects of any
bubbles that may form in the pumps, valves, or tubing. In some
embodiments, microsensors or biosensors can also be integrated into
the organ chips and/or in vitro microphysiological systems for
controlling the culture conditions and/or monitoring the response
of cells to the culture conditions. Any art-recognized biosensors,
e.g., thin enzyme electrodes (Ref. 5) and/or microphysiometers
(Ref. 6) can be used in any embodiments of the organ chips and/or
in vitro microphysiological systems described herein.
[0066] In some embodiments of any types of organ chips described
herein, organ chips can be oxygenated either through a porous
material used in the construction of the organ chips (e.g., PDMS)
or using on-cartridge or systemic gas exchange membranes.
[0067] An organ chip can be produced with or without aerosol
delivery capabilities. In some embodiments, an organ chip can be
adapted to deliver an aerosol, e.g., comprising an active agent
described herein, to cells cultured in the channels. Detailed
information about various designs and configurations of
microfluidic devices for aerosol delivery can be found, e.g., in
the International Application No. WO 2012/154834, the contents of
which are incorporated herein by reference in their entireties.
[0068] As an organ chip is developed to mimic the respective
function of an organ, the design of each organ chip can be
different according to their respective physiological properties
and/or functions. For example, the organ chips can differ in, e.g.,
but not limited to, cell populations (e.g., cell types and/or
initial cell seeding density), internal design, microarchitecture,
dimensions, fluidic control, mechanical and electrical control and
read-outs depending on the organ type (e.g., Lung Chip versus Heart
Chip).
[0069] In some embodiments, the organ chips can be designed to have
a common shape and have positioned inlets and outlets for delivery
of fluids to the Microvascular channels lined by microvascular
endothelium and Interstitial fluid channels lined by organ-specific
parenchymal cells (e.g., but not limited to, alveolar epithelium,
heart muscle, hepatocytes). See, e.g., the section ""Examples of
organ chips or organ-on-a-chip devices" for exemplary design of
various organ chips based in part on the two basic organ chips
(e.g., Lung chips and Heart chips) described herein.
[0070] Without limitations, different kinds of organ chips
described herein can comprise additional cell types, e.g., but not
limited to, immune cells, stromal cells, smooth muscle cells,
neurons, lymphatic cells, adipose cells, and/or microbiome in gut,
based on the goals of the application. By way of example only, if
inflammatory response is desired to be studied in a gut or liver
model, immune cells can be incorporated into the gut or liver chip
accordingly.
[0071] Functional Assessment of Organ Chips:
[0072] The viability and/or function of various organ chips can be
generally assessed, e.g., morphologically with optical imaging. In
some embodiments, any other art-recognized characterization
techniques can be used to determine the function of various organ
chips. For example, the alveolar-capillary interface function of
the Lung Chip can be measured, e.g., by quantifying permeability
barrier function (e.g., using TEER and molecular exclusion),
measuring surfactant production, and/or demonstrating physiological
relevant responses to cytokines (e.g., ICAM1 expression in response
to TNF.alpha.). See e.g., Huh D. et al., 2010. Heart muscle
function can be characterized, e.g., using force-frequency curves,
measuring increases in peak contraction stress as a function of
increasing field stimulation frequency, and/or analyzing
electrocardiogram results during the same protocol to ensure that
the tissues are functioning electrically. See Grosberg A. et al.
2011. Functionality of the Liver Chip can be assessed, e.g., via
multiple well established assays including albumin secretion,
transporter expression and/or function (efflux and uptake
transporters), and/or CYP450 expression. Specific CYP450 enzyme can
be determined, e.g., by incubation with FDA approved probe
substrates REF1, and specific metabolite formation for each CYP450
isoform can be measured and validated, e.g., using LC/MS. Response
of hepatocytes to prototypical CYP450 inducers (e.g., Rifampacin
for CYP3A4) can be assessed.
[0073] Based on the functional assessments, one of skill in the art
can adjust the condition of the organ chips, e.g., by modulating
the flow rate of fluid (fluid shear stress), nutrient level,
mechanical stimulation, electrical stimulation, cell seeding
density on the membranes, cell types, ECM composition on the
membrane, dimension and/or shapes of the channels, oxygen gradient
and any combinations thereof, to modulate the functional outcome of
the organ chips, or the in vitro microphysiological system.
Parenchymal Cells and Vascular Endothelial Cells
[0074] Parenchymal cells are selected to suit for specific organ
chips. Parenchymal cells are generally the distinct cells of an
organ contained in and supported by the connective tissue
framework. The parenchymal cells typically perform a function that
is unique to the particular organ. In some embodiments, the term
"parenchymal" can exclude cells that are common to many organs and
tissues such as fibroblasts and endothelial cells within blood
vessels.
[0075] For example, in a liver organ, the parenchymal cells can
include hepatocytes, Kupffer cells, epithelial cells that line the
biliary tract and bile ductules, and any combinations thereof. The
major constituent of the liver parenchyma are polyhedral
hepatocytes (also known as hepatic cells) that presents at least
one side to an hepatic sinusoid and opposed sides to a bile
canaliculus. Liver cells that are not parenchymal cells include
cells within the blood vessels such as the endothelial cells or
fibroblast cells.
[0076] In striated muscle, the parenchymal cells can include
myoblasts, satellite cells, myotubules, myofibers, and any
combinations thereof.
[0077] In cardiac muscle, the parenchymal cells can include the
myocardium also known as cardiac muscle fibers or cardiac muscle
cells, the cells of the impulse connecting system such as those
that constitute the sinoatrial node, atrioventricular node,
atrioventricular bundle, and any combinations thereof.
[0078] In a pancreas, the parenchymal cells can include cells
within the acini such as zymogenic cells, centroacinar cells, basal
or basket cells, cells within the islets of Langerhans such as
alpha and beta cells, and any combinations thereof.
[0079] In spleen, thymus, lymph nodes and bone marrow, the
parenchymal cells can include reticular cells, blood cells (or
precursors to blood cells) such as lymphocytes, monocytes, plasma
cells, macrophages, and any combinations thereof.
[0080] In the kidney, parenchymal cells can include cells of
collecting tubules, the proximal and distal tubular cells, and any
combinations thereof.
[0081] In the prostate, the parenchyma can include epithelial
cells.
[0082] In glandular tissues and organs, the parenchymal cells can
include cells that produce hormones. In the parathyroid glands, the
parenchymal cells can include the principal cells (chief cells),
oxyphilic cells, and a combination thereof. In the thyroid gland,
the parenchymal cells can include follicular epithelial cells,
parafollicular cells, and a combination thereof. In the adrenal
glands, the parenchymal cells can include the epithelial cells
within the adrenal cortex and the polyhedral cells within the
adrenal medulla.
[0083] In the parenchyma of the gastrointestinal tract such as the
esophagus, stomach, and intestines, the parenchymal cells can
include epithelial cells, glandular cells, basal cells, goblet
cells, and any combinations thereof.
[0084] In the parenchyma of lung, the parenchymal cells can include
the epithelial cells, mucus cells, goblet cells, alveolar cells,
and any combinations thereof.
[0085] In the skin, the parenchymal cells can include the
epithelial cells of the epidermis, melanocytes, cells of the sweat
glands, cells of the hair root, and any combinations thereof.
[0086] Cell Sources:
[0087] The cells (e.g., parenchymal cells and/or vascular
endothelial cells) used in the organ chips can be isolated from a
tissue or a fluid of subject using any methods known in the art, or
differentiated from stems cells, e.g., embryonic stem cells, or
iPSC cells, or directly differentiated from somatic cells. In some
embodiments, stem cells can be cultured inside the organ chips and
be induced to differentiate to organ-specific cells. Alternatively,
the cells used in the organ chips can be obtained from commercial
sources, e.g., Cellular Dynamics International, Axiogenesis,
Gigacyte, Biopredic, InVitrogen, Lonza, Clonetics, CDI, and
Millipore, etc.).
[0088] In some embodiments, the cells used in the organ chips can
be differentiated from the "established" cell lines that commonly
exhibit poor differentiated properties (e.g., A549, CaCo2, HT29,
etc.). These "established" cell lines can exhibit high levels of
differentiation if presented with the relevant physical
microenvironment (e.g., air-liquid interface and cyclic strain in
lung, flow and cyclic strain in gut, etc.), e.g., in some
embodiments of the organ chips.
[0089] In some embodiments, the cells used in the organ chips can
be genetically engineered for various purposes, e.g., to express a
fluorescent protein, or to modulate an expression of a gene, or to
be sensitive to an external stimulus, e.g., light, pH, temperature
and/or any combinations thereof.
Examples of Organ Chips or Organ-On-a-Chip Devices
[0090] An in vitro microphysiological system can comprise at least
two different organ chips using one or both of the first and second
organ chip designs described herein. The first organ chip design is
based a microfluidic device comprising: a body comprising a central
channel therein, and an least partially porous and at least
partially flexible first membrane positioned within the central
channel and along a plane, wherein the first membrane is configured
to separate the central channel to form two sub-channels, wherein
one side of the first membrane is seeded with vascular endothelial
cells, and the other side of the first membrane is seeded with at
least one type of organ-specific parenchymal cells.
[0091] In some embodiments, the first organ chip can further
comprise at least a channel wall positioned adjacent to the two
sub-channels, wherein the first membrane can be mounted to the
channel wall; and an operating channel adjacent to the two
sub-channels on an opposing side of the channel wall, wherein a
pressure differential applied between the operating channel and the
two sub-channels can cause the channel wall to flex in a desired
direction to expand or contract along the plane within the two
sub-channels.
[0092] The second organ chip design is based on a microfluidic
device comprising: a body comprising a first chamber enclosing a
plurality of muscular thin films adapted to measure contraction of
muscle cells, and a second chamber comprising a layer of muscle
cells on the bottom surface of the second chamber, wherein the
bottom surface is embedded with an array of microelectrodes for
recording of action potentials, and wherein the top surface of the
second chamber is placed with at least a pair of electrodes for
providing electric field stimulation to the muscle cells.
[0093] In some embodiments, the second organ chip can further
comprise an at least partially porous second membrane positioned
within the first chamber to form a top chamber and a bottom
chamber, wherein the bottom chamber can comprise the plurality of
muscular thin films on its bottom surface, and wherein the surface
of the second membrane in contact with the top chamber can be
seeded with a layer of epithelial cells.
[0094] The design of the first organ chip described herein can
provide a basis for development of organ chips to mimic
tissue-tissue interfaces, and/or mechanically-active
microenvironment. For example, lung chips, liver chips, gut chips,
kidney chips, skin chips, brain chips, testis chips, and any
combinations thereof, can be constructed based on the first organ
chip design with any appropriate modifications.
[0095] The design of the second organ chip described herein can
provide the basis for development of organ chips where cell
contraction, and/or electric field stimulation of the cells are
intended. For example, heart chips, skeletal muscle chips, lung
airway smooth muscle chips, brain chips, and any combinations
thereof can be constructed based on the second organ chip design
with any appropriate modifications.
[0096] In some embodiments, an organ chip, e.g., a brain chip can
employ either or both designs of the first and second organ chips,
with any appropriate modifications.
[0097] The following examples of organ chips are intended to
illustrate applications and/or adaptations of the first organ chip
design and/or second organ chip design to construct various organ
chips, and should not be construed to be limiting. Any
modifications to the organ chips described herein that are within
one of skill in the art are also encompassed by the scope described
herein.
[0098] Lung Chips or Lung-On-a-Chip:
[0099] The methods, multi-channeled architecture and ability of a
Lung Chip (FIG. 1 and FIGS. 2A-2B) to mimic, at least in part, the
normal physiology (e.g., normal breathing) of a lung are based on
the first organ chip design and has been previously described in
Huh D. et al. "Reconstituting organ-level lung function on a chip"
Science (2010) 328: 1662, and in the International Application No.
WO 2010/009307, the contents of which are incorporated herein by
reference in their entireties.
[0100] For example, FIGS. 2A-2B shows diagrammatic views of a
lung-on-a-chip in accordance with one embodiment described herein.
The lung chip can comprise a body 202 having a central microchannel
204 therein; and an at least partially porous and at least
partially flexible membrane 206 positioned within the central
microchannel 204 and along a plane. The membrane 206 is configured
to separate the central microchannel 204 to form a first central
microchannel 208 and a second central microchannel 210, wherein a
first fluid is applied through the first central microchannel 208
and a second fluid is applied through the second central
microchannel 210. There is at least one operating channel (212A,
212B) separated from the first 208 and second 210 central
microchannels by a first microchannel wall 214. The membrane 206 is
mounted to the first microchannel wall 214, and when a pressure is
applied to the operating channel (212A and/or 212B), it can cause
the membrane to expand or contract along the plane within the first
208 and the second 210 central microchannels.
[0101] In some embodiments, one side of the membrane 206 can be
seeded with alveolar epithelial cells to mimic an epithelial layer
while another side of the membrane 206 can be seeded with lung
microvascular endothelial cells to mimic capillary vessels.
Accordingly, lung chips, in some embodiments can be used to mimic
an alveolar-capillary unit, which 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.
[0102] In such embodiments, a gaseous fluid, e.g., air and/or
aerosol, can flow through the first central microchannel 208 in
which the alveolar epithelial cells are resided, while a liquid
fluid, e.g., culture medium, buffered solution and/or blood, can
flow through the second central microchannel 210 (Microvascular
channel) in which the microvascular endothelial cells are
resided.
[0103] The lung chips can be used to evaluate lung response to an
active agent, e.g., but not limited to, immune response to
microbial infection and/or inflammatory responses to
nanoparticulate toxins. For example, the active agent (e.g., cells
including, e.g., but not limited to, bacteria and/or virus,
proteins, peptides, antigens, antibodies or portions thereof,
enzymes, nucleic acids, siRNA, shRNA, aptamers, small molecules,
antibiotics, drugs or therapeutic agents, molecular toxins,
nanomaterials or particulates, aerosols, environmental contaminants
or pollutants (e.g., but not limited to, microorganisms,
organic/inorganic contaminants present in food and/or water, and/or
air pollutants), and any combinations thereof) can be added in a
liquid fluid flowing through the second central microchannel 210,
e.g., to mimic blood carrying the active agent (e.g., cells
including, e.g., but not limited to, bacteria and/or virus,
proteins, peptides, antigens, antibodies or portions thereof,
enzymes, nucleic acids, siRNA, shRNA, aptamers, small molecules,
antibiotics, drugs or therapeutic agents, molecular toxins,
nanomaterials or particulates, aerosols, environmental contaminants
or pollutants (e.g., but not limited to, microorganisms,
organic/inorganic contaminants present in food and/or water, and/or
air pollutants), and any combinations thereof). The inventors have
demonstrated, in one embodiment, that the lung chips that mimic the
lung's dynamic mechanically-active (breathing) microenvironment
(e.g., using the device described in the International Application
No. WO 2010/009307 in which one embodiment has side channels to
allow modulation of pressure to cause cyclic movement of the
flexible porous membrane on which the cells are seeded) can
effectively predict lung toxicity responses to the chemotherapeutic
cytokine, e.g., IL-2, which has a dose-limiting toxicity due to
vascular leakage leading to pulmonary edema in humans. Using the
lung-on-a-chip with a detectable marker, e.g., fluorescent-insulin
as a marker of vascular permeability (fluid shifts), the IL-2
produces a small but significant increase in pulmonary vascular
leakage into the air channel of the lung chip under static
conditions. However, with physiological breathing motions akin to
normal breathing motions (10% cyclic strain), this response is
increased by more than 3-fold (and it was accompanied by blood clot
formation as seen in humans), and the critical physiological
importance of providing this correct mechanical microenvironment
was demonstrated in studies in a mouse ex vivo
ventilation-perfusion model that demonstrated a similar dependency
of pulmonary edema induction by IL2 on breathing motions (FIG. 3).
Using the lung chips, various kinds of drugs or candidate agents
can be tested to determine what would be the effective treatment.
In fact, the IL2-induced pulmonary toxicity in the lung chip can be
pharmaceutically suppressed in vitro. These results provide
proof-of-principle for organ chips lined by human cells (e.g.,
organ-specific parenchymal cells) as a means to predict clinically
relevant toxicity responses in humans.
[0104] In some embodiments, the microchannel intended for a gaseous
flow, e.g., the first central microchannel 208 allowing air to flow
through, can be configured to permit delivery of aerosolized
micro-droplets (e.g., aerosolized drugs, and/or nanoparticles or
particulates). Detailed information about various designs and
configurations of microfluidic devices for aerosol delivery can be
found, e.g., in the International Application No. WO 2012/154834,
the content of which is incorporated herein by reference in its
entirety, and can be adopted in the lung chips to deliver
aerosolized microdroplets, e.g., to study toxicities of
nanoparticles in lungs. Similar findings in relation to the
toxicities of nanoparticles has been observed using the lung chips,
indicating that some clinically organ toxicities cannot be mimicked
in vitro without providing the correct mechanical microenvironment,
which is generally lacking from traditional in vitro model
systems.
[0105] Heart Chips:
[0106] The methods, architecture and ability of a Heart Chip (e.g.,
FIG. 8) to mimic, at least in part, the normal physiology (e.g.,
cell contraction) of a heart are based on the second organ chip
design described herein. Heart Chips are described, for example, in
U.S. Provisional Patent Application Ser. No. 61/569,028, filed on
Dec. 9, 2011, U.S. Provisional Patent Application Ser. No.
61/697,121, filed on Sep. 5, 2012, and PCT patent application
titled "Muscle Chips and Methods of Use Thereof," filed on Dec. 10,
2012 and which claims priority to the U.S. provisional application
No. 61/569,028, filed on Dec. 9, 2011, U.S. Provisional Patent
Application Ser. No. 61/697,121, the entire contents of all of
which are incorporated herein by reference in their entireties.
[0107] In order to fabricate heart chips to mimic tissue
organization, in some embodiments, functional heart tissues can be
fabricated first and then multiplexed in a microfluidic device. For
example, functional heart tissues can be fabricated by culturing
ventricular cardiomyocytes (e.g., neonatal rat ventricular
cardiomyocytes) on elastomeric polymer thin films micropatterned
with cell adhesion proteins (e.g., extracellular matrix proteins)
to promote spatially ordered, two-dimensional myogenesis and create
"muscular thin films" (MTFs) as described previously, e.g., in
Grosberg A. et al. "Ensembles of engineered cardiac tissues for
physiological and pharmacological study: Heart on a chip" Lab on a
chip (2011) 11: 4165, as well as the International Application Nos.
WO 2008/045506, WO 2010/011407 and WO2010/042856, the contents of
which are incorporated herein by reference in their entireties.
These heart tissue constructs are electrically functional and
actively contractile, generating stresses comparable to those
produced by whole papillary muscle.
[0108] As used herein, the terms "muscular thin film" and "MTF" are
used interchangeably herein and refer to a two-dimensional
biopolymer substrate comprising heart muscle cells such as
ventricular cardiomyocytes or progenitor cells, which can cause the
substrate to bend and form a three-dimensional (3D) structure when
the cells contract, e.g., as shown in FIG. 6 or FIG. 7. The MTFs
(e.g., at least about 2, at least about 3, at least about 4, at
least about 5, at least about 6, at least about 7, at least about
8, at least about 9, at least about 10, at least about 20, at least
about 30, at least about 40, at least about 50 or more MTFs) can
then be multiplexed, e.g., in an array, within a microfluidic chip
(FIGS. 4B-4C). The MTFs can be used to measure effects on heart
cell contractile function in vitro during electrical and
pharmacological stimulation (Grosberg et al., 2011, FIG. 5).
[0109] In another embodiment, multi-layered heart chips can be
constructed. For example, a multi-layered heart chip can comprise a
body having a `Microvascular Channel` lined by endothelium (e.g.,
human endothelial cells) adherent to a porous membrane that
separates the Microvascular Channel from the MTF-lined
`Interstitial Channel`, such as similar to the configuration of the
lung chip as shown in FIG. 2, but without side chambers. The
inventors have demonstrated that microengineered MTFs effectively
mimic pharmacological responses of adult rat papillary muscle
strips (FIG. 5), which are commonly used to screen cardiac tissue
responses to drugs by the pharmaceutical industry.
[0110] In some embodiments, the heart chips can be modified for
various analyses. For example, as shown in FIG. 8, the heart chips
800 can have at least one set of MTF (804)-lined Interstitial
Channels (including at least 2 sets, at least 3 sets, at least 4
sets, at least 5 sets or more) to form a MTF Chamber 802, e.g., for
optical imaging and contractility analysis of cells exposed to a
culture medium with or without an active agent described herein,
e.g., by optical measurements of radius of curvatures or projected
length of MTFs using microscopy. By way of example only, a curled
or contracted MTF can have a smaller projected length than a
relaxed MTF, e.g., as shown in FIG. 8 (iv).
[0111] To create a MTF chamber, by way of example only, a laser
engraving process, e.g., as shown in FIG. 6, can be utilized to cut
PDMS thin films of cardiomyocytes cultured as described above;
followed by microcontact-printing ECM proteins and enclosing the
assembly in a microfluidic chamber. Alternatively, the thin films
of cardiomyocytes can be produced with alginate (FIG. 7). To
operate a MTF chamber, muscle cells can be cultured on the thin
films and the contractile stresses can be assessed by optically
monitoring the extent of curvature of the muscular thin films as
noted above (FIG. 8). An alternative strategy for measuring
contractility is depicted in FIG. 9, where instead of making MTFs,
the muscle cells can be grown on a wrinkling substrate 902, e.g., a
polyurethane membrane, such that as the muscle cells contract, the
substrate deforms. In this embodiment, the wrinkling substrate can
further comprise a plurality of holes 904 (e.g., a swiss
cheese-like polyurethane membrane) to facilitate optical detection
of the substrate deformation due to muscle cell contraction. For
example, the holes within the substrate can remain as a circle when
the muscle cells are relaxed, but the circle becomes deformed,
e.g., becoming an oval, or an ellipse, due to muscle cell
contraction.
[0112] The dimensions of the MTF chamber can be suited for a user's
need. For example, the length of the MTF chamber can vary from
about 5 mm to about 100 mm, or from about 10 mm to about 50 mm, or
from about 10 mm to about 25 mm. In one embodiment, the length of
the MTF chamber can be about 15 mm. The height of the MTF chamber
can be adjusted, e.g., based on the size of MTFs. For example, the
height of the MTF chamber is designed to be sufficient to permit
the MTFs to freely bend or curl up without any spatial constraint.
In some embodiments, the height of the MTF chamber can be about 0.5
mm to about 5 mm, or about 0.5 mm to about 3 mm. In one embodiment,
the height of the MTF chamber can be about 1 mm.
[0113] In some embodiments, the heart chips, e.g., the Interstitial
Channels can further comprise any heart-specific parenchymal cells,
e.g., but not limited to cardiomyocytes and/or vascular smooth
muscle cells, to further mimic the physiological environment and/or
function of the heart. In some embodiments, a heart chip can be a
microfluidic device in which mature cardiomyocytes (e.g., human
cardiomyocytes) are cultured on a 2D polymer substrate (e.g., PDMS
substrates or micromolded alginate substrates) to form MTFs. In
some embodiments, the heart chips can have both vascular smooth
muscle and cardiac muscle cultured on the same chip (see FIG. 10),
e.g., to form to different kinds of muscles, e.g., smooth muscles
vs. striated muscles.
[0114] The cardiomyocytes can be isolated from a tissue of a
subject or obtained from a commercial source, or by differentiating
stems cells to cardiomyocytes, e.g., induced pluripotent stem
cell-derived cardiomyocytes. Cardiomyocytes (e.g., human
cardiomyocytes) can be obtained from a commercial source, e.g.,
from Axiogenesis, CDI, Vistagen, Coriell, Reprocell. The culture
conditions can be optimized for each source of cardiac myocytes,
e.g., ES- or iPS-derived cardiac myocytes, neonatal rat or mouse
myocytes. The human cardiomyocytes can be obtained or derived from
different origins (e.g., ventricular, atrial, etc.). In some
embodiments, the cardiomyocytes of different origins can be
co-cultured. In some embodiments, the cardiomyocytes can be
engineered to be photosensitive (e.g., cardiomyocytes expressing a
photosensitive membrane transport mechanism that is responsive to
light of a particular wavelength). See, e.g., the International
Appl. No. WO 2012/006320, the content of which is incorporated
herein by reference in its entirety, for information about
fabrication of photosensitive cardiac rhythm modulation systems,
which can be integrated into the heart chips described herein.
[0115] To fabricate an in vitro myocardial construct that
recapitulates the structural complexity of the human heart
chambers, in one embodiment, aligned or anisotropic monolayers of
muscle cells (e.g., a monolayer of cardiomyocytes with a laminar
organization) can be cultured on microcontact printed biocompatible
substrates (e.g., PDMS substrates, microcontact printed
polyurethane membranes and micromolded alginate substrates) within
a microfluidic device, e.g., being integrated into a MTF chamber
for contractility measurements and/or a low volume
Electrophysiological Chamber (or an Electrophysiological Chamber)
as described below.
[0116] In some embodiments, the heart chips 800 can further have at
least one another set of Interstitial Channels (including at least
2 sets, at least 3 sets, at least 4 sets, at least 5 sets or more)
lined with a heart muscle cell layer under which are embedded with
at least one or a plurality (e.g., an array) of microelectrodes
(e.g., platinum microelectrodes) 808 (microelectrodes are placed
underneath the cells), while the side of the channel opposing the
heart muscle cell layer is placed with at least one or more (e.g.,
at least two or more) electric field stimulation electrodes 810 to
form an "Electrophysiological Chamber" 806 (see, e.g., FIG. 8). In
such embodiments, the Electrophysiological Chamber 806 can further
comprise an electrocardiography (ECG) lead port 812 for connection
with an ECG lead (or a lead electrocardiogram) to measure and/or
monitor electrical pacing and/or analysis of changes in cardiac
electrical potential.
[0117] To fabricate an Electrophysiological (EPhys) Chamber 806, an
anisotropic muscle cell monolayer can be cultured in a low volume
chamber. Then, the muscle cells can be electrically field
stimulated using the electrodes 810 on the top, and action
potentials can be recorded using the microelectrode array 808 on
the bottom, where the microelectrode array is placed underneath the
muscle cells cultured on the bottom surface. The electrode readouts
can be calibrated with the action potential characteristics of the
cardiomyocyte monolayer.
[0118] The dimensions of the Electrophysiological Chamber can be
suited for a user's need. For example, the length of the
Electrophysiological Chamber can vary from about 5 mm to about 100
mm, or from about 8 mm to about 50 mm, or from about 10 mm to about
25 mm. In one embodiment, the length of the Electrophysiological
Chamber can be about 10 mm. The height of the MTF chamber can be
adjusted, e.g., based on the size of field stimulation electrodes.
For example, the height of the MTF chamber is designed to be
sufficient to permit the MTFs to freely bend or curl up without any
spatial constraint. In some embodiments, the height of the MTF
chamber can be about 0.5 mm to about 5 mm, or about 0.5 mm to about
3 mm. In one embodiment, the height of the MTF chamber can be about
1 mm.
[0119] The electrophysiological chamber 806 can be placed anywhere
relative to the MTF chamber 802. In some embodiments, the
electrophysiological chamber 806 can be placed in parallel to the
MTF chamber 802. In some embodiments, the MTF chamber can be larger
than the Electrophysiological Chamber to permit high throughput
contractility measurements.
[0120] While the Electrophysiological 806 and MTF 802 Chambers can
each be independently fed by a separate culture medium stream, in
some embodiments, both the Interstitial Channels in the MTF and
Electrophysiological Chambers can be fed by a single medium stream
814. In some embodiments, the culture medium can be introduced
directly through the Interstitial Channels in the
Electrophysiological and MTF chambers and/or introduced through an
underlying endothelium-lined Microvascular Channels, e.g., in a
configuration similar to the lung chips (see, for example, FIG. 2).
The presence of the endothelium and its basement membrane lining
the microvascular channel on the opposite side of the membrane,
plus the ability to perfuse different media compositions through
the Interstitial versus Microvascular Channels can allow different
experimental conditions for various applications.
[0121] In some embodiments, the heart chips can comprise a MTF
chamber and an EPhys chamber situated next to each other, for
example, as shown in FIG. 8.
[0122] In some embodiments, the heart chip can be adapted to
fluidically connected upstream and/or downstream to at least one or
more organ chips (e.g., but not limited to lung chips or liver
chips).
[0123] Prediction/Determination and Validation of Pharmacological
Effects:
[0124] The heart chips described herein can be used to determine
pharmacological effects on heart-specific cells cultured therein.
For example, the heart-specific cells can be exposed to an active
agent (e.g., cells including, e.g., but not limited to, bacteria
and/or virus, proteins, peptides, antigens, antibodies or portions
thereof, enzymes, nucleic acids, siRNA, shRNA, aptamers, small
molecules, antibiotics, drugs or therapeutic agents, molecular
toxins, nanomaterials or particulates, aerosols, environmental
contaminants or pollutants (e.g., but not limited to,
microorganisms, organic/inorganic contaminants present in food
and/or water, and/or air pollutants), and any combinations thereof)
by adding the active agent in a culture medium flowing through the
Interstitial Channels and/or Microvascular Channels. In some
embodiments where the heart chip comprises an Electrophysiological
Chamber, electrophysiological information can be collected within
the chip, which can be in turn modeled with a user-defined
algorithm to estimate an ECG. The ECG can then be correlated with
reference data (e.g., current in vitro and outputs, or control
outputs, e.g., outputs of cells not exposed to the active agent).
In one embodiment, the radius of curvature of the contracting MTFs
within the chip can be modeled to estimate the stresses exerted by
the cells. The MTF contractility measurements can then be
correlated with reference data (e.g., current in vitro and clinical
outputs, or control outputs, e.g., outputs of cells not exposed to
the active agent).
[0125] To validate the heart chips for assessment of
pharmacological effects, known drugs within the system can be used,
such that the response of the heart chips can be correlated with
clinical response to the known drugs. In some embodiments, the
cardiac myocytes inside the heart chips can be exposed to drugs
that are known to be cardio-safe (e.g., ibuprofen and
fexofenadine), e.g., to show that they have no toxic effects on
engineered myocardial tissue constructs. In some embodiments, the
cardiac myocytes inside the heart chips can be exposed to drugs
that are cardiotoxic (e.g., doxorubicin, trastuzumab, terfenadine).
For example, doxorubicin (anthracycline family of
chemotherapeutics) generally displays cardiotoxic effects primarily
by inducing dilated cardiomyopathy, predominately affect the
ventricles with some left atrial involvement. Trastuzumab is a
monoclonal antibody to HER2 receptor used for the treatment of
(HER2+) breast cancers, has been shown to decrease left ventricular
ejection fraction. Terfenadine is antihistamine formerly used to
treat allergic conditions that was found to be cardiotoxic because
it binds hERG channels and causes long QT syndrome. Sorafenib and
sunitinib are tyrosine kinase inhibitors used as chemotherapeutic
agents that cause poorly characterized cardiotoxic effects that
usually manifest as decreased left ventricular ejection fraction
and increased cardiac enzymes.
[0126] Exemplary Applications of Heart Chips:
[0127] Although various drug toxicities can affect myocytes with
distinct mechanisms, most of the drugs can adversely affect cardiac
contractility and ultimately the ability of the heart to pump
blood. Therefore, by measuring the contractility and/or
electrophysiological output of the heart chip, one can
quantitatively measure the impact of various active agents on heart
performance. Further, the chambers of the heart are imperative to
its function, as the ventricles of the heart are tasked with
overcoming pulmonary and system vascular resistance and after load.
For example, drugs such as doxorubicin and trastuzumab (as noted
above) generally demonstrate ventricular toxicity; thus, some
embodiments of the heart chips described herein can be used to
mimic the contractile action of these chambers (e.g., ventricular
chambers) in vitro. In some embodiments, the heart chip can be a
microfluidic device that can measure electrophysiology and/or
contractile performance of engineered myocardial tissues. In some
embodiments, the heart chip can be a microfluidic device that can
measure electrophysiology and/or contractile performance of
myocardial tissues derived or obtained from a subject, e.g., to
determine an appropriate treatment regimen for the specific
subject.
[0128] In some embodiments, the heart chips can be used as disease
models, e. g., to assess different physiological effects of
pharmaceutical or active agents. Examples of the effects that can
be assessed using the heart chips can include, without limitations,
(1) metabolic: for example, antibody-based medications such as
Trastuzumab, which is generally used to treat HER2+ breast cancer,
are known to affect ATP production in myocytes through adverse
effects on mitochondria (Ref. 7); (2) structural: for example,
anti-proliferative agents such as doxorubicin, which is used for a
wide variety of malignancies, can adversely affect myocyte growth
and mass maintenance (Ref. 8); and (3) ion channel
(arrhythmia-inducing): for example, hERG channel binding of many
drugs (e.g. albuterol) can generally increase action potential
duration, leaving the heart susceptible to arrhythmia (long QT
syndrome) and effects on other ion channels (Na, Ca), with
concurrent effects on PR prolongation, conduction abnormalities and
arrhythmias. While there are in vitro models exist for hERG channel
binding assays (Ref. 9), there is a lack of in vitro assays for
evaluating drugs exerting metabolic and/or structural effects that
can deliver quantitative data relating to cardiac function.
Accordingly, a robust in vitro system that can recapitulate the
heart can provide a platform to screen a large number of compounds
for toxicity, a feat not possible with current in vivo animal
models or existing in vitro models.
[0129] Since tissue architecture can be controlled within heart
chips, which can in turn affect contractile efficiency, various
disease states can be created accordingly. Drug toxicity (e.g.,
Trastuzumab or Doxorubicin) can affect the ability of cardiac
myocyte contractility. Thus, in one embodiment, drug toxicity can
be determined by measuring contractility of cardiac myocytes within
the heart chips. For example, contractility can be read out via
optical tracking of MTF deformation/optical tracking of membrane
deformation as described above. Optionally, electrophysiological
recordings can be taken via a lead ECG. In some embodiments,
biomarkers of muscle damage such as creatine kinase and troponin
can also be measured. For example, the level of biomarkers can be
measured in the culture medium upon exposure to the cells.
[0130] In some embodiments, the heart chips can be used for PK/PD
modeling (including prediction of pharmacological effects).
[0131] Generally, the design of other organ chips can be developed
based on the basic designs of the Lung or Heart chips as described
herein. For example, without being construed to be limiting, Gut
chips, Kidney chips, Liver chips, Skin chips and Testis chips can
be developed, e.g., based on the basic design of the Lung chips,
while Skeletal Muscle chips, Airways Smooth Muscle chips can be
developed, e.g., based on the design of the Heart chips. Depending
on different organs, each organ chip can then be incorporated with
respective tissue-specific parenchymal cell (e.g., human
parenchymal cell) layers within the Interstitial Channel (as
described earlier) exposed to their physiological microenvironment
(e.g., alveolar epithelium and skin epidermis exposed to air, gut
epithelium facing a fluid filled lumen, etc.) on one surface of the
ECM-coated porous membrane, with organ-specific vascular
endothelium (e.g., human vascular endothelium) on the opposite side
lining the Microvascular Channel. Organ-specific differences in the
mechanical microenvironment can also be mimicked by altering
control and/or process parameters, e.g., but not limited to, flow
rates, fluid shear stresses, cyclic mechanical strain, ECM
composition, and/or compartment dimensions.
[0132] Liver Chips:
[0133] In some embodiments, a mammalian liver chip (e.g., a human
liver chip) can be developed and/or modified, e.g., from the basic
Lung Chip multichannel design described herein (e.g., as shown in
FIG. 2) as well as in Huh D. et al., 2010 and/or in the
International Application No. WO 2010/009307, the contents of which
are incorporated herein by reference in its entirety. For example,
commercially available human hepatocytes (e.g., from Invitrogen) or
patient-specific hepatocytes (isolated from a tissue) can be placed
on one side of the ECM (e.g., but not limited to, laminin, type IV
collagen or Matrigel)-coated membrane, and human microvascular
endothelial cells on the other side. Without wishing to be bound by
theory, the porous membrane, basement membrane and cell-cell
junctions of the endothelium can facilitate physiologically
relevant mass transport while protecting hepatocytes from fluid
shear stress and serum components in the Microvascular Channel.
[0134] In some embodiments, the liver chips can further comprise
other parenchymal cells, such as Kupffer cells (resident
macrophages of the liver) under the endothelium in the Liver
Chip.
[0135] Without wishing to be bound by theory, oxygen gradients are
essential determinants of normal liver physiology and function, as
well as key contributors to acute and chronic hepatoxicity.
Accordingly, in some embodiments, oxygen gradients found in vivo
can be incorporated in the liver chips. By way of example only,
oxygen gradients can be generated in a liver chip, e.g., by using a
gas-permeable membrane material, e.g., PDMS which is permeable to
gases) and flowing oxygen at different concentrations through the
two side channels separated by the membrane. Other art-recognized
microengineering methods, for example, as described in Adler M. et
al. Lab Chip 2010; 10(3): 388-389 and Chen Y-A et al., Lab Chip
2011; 11: 3626-3633, can also be used to develop oxygen gradients
within the organ chips.
[0136] In some embodiments, bile canalicular networks in
pre-determined patterns (e.g., by art-recognized micropatterning
techniques) can be integrated into liver chips, such that they can
be coupled with microscale sampling ports in the liver chips. Such
configuration can be used to determine intrinsic biliary clearance.
This approach can be used to facilitate analysis of interplay
between drug transporters and drug metabolizing enzymes, which is a
key determinant of drug PK properties and toxicity profiles in
humans.
[0137] Gut Chips:
[0138] In some embodiments, the gut chips, e.g., as shown in FIG.
11, can be developed and/or modified, e.g., from the basic Lung
Chip multichannel design described herein (e.g., as shown in FIG.
2) as well as in Huh D. et al., 2010 and/or in the International
Application No. WO 2010/009307, the contents of which are
incorporated herein by reference in their entireties. In some
embodiments, a gut chip can utilize a porous membrane (e.g., PDMS
membrane) coated with ECM (e.g., Collagen I+Matrigel) upon one side
of which human intestinal epithelial cells are cultured under flow
conditions to produce a physiological shear stress (.about.0.02
dyne/cm.sup.2) while simultaneously exerting cyclic mechanical
strain (10% elongation, 0.15 Hz). Gut-specific microvascular
endothelial cells can be seeded and cultured on another side of the
porous membrane to form a Microvascular Channel as described above.
For example, human gut cells (e.g., CaCo2 cells) cultured under
these conditions can differentiate by changing their entire
transcriptome (measured, e.g., using gene microarrays) and form 3D
villus structures, e.g., to match the height of the microfluidic
Interstitial Channel (FIG. 11F). In addition, the physiologically
relevant conditions recreated in the Gut Chip can enable one to
culture living gut bacteria (Lactobacillus) directly on top of the
living human villus gut epithelium, and hence, permit interrogating
the influence of gut microbiome on drug absorption and metabolism
using the gut chips.
[0139] In some embodiments, the gut chips described herein can
comprise any components of the device or can be the device
described in the International Appl. No. WO 2012/118799, the
content of which is incorporated herein by reference in its
entirety.
[0140] Kidney Chips:
[0141] Without limitations, in some embodiments, the kidney chips
can be developed and/or modified, e.g., from the basic Lung Chip
multichannel design described herein (e.g., as shown in FIG. 2) as
well as in Huh D. et al., 2010 and/or in the International
Application No. WO 2010/009307, the contents of which are
incorporated herein by reference in their entireties. In some
embodiments, the kidney chips can utilize a porous membrane (e.g.,
a PDMS membrane) coated with ECM (e.g., type IV collagen) upon one
side of which primary human proximal tubular epithelial cells
(e.g., obtained from Biopredic or from a specific subject) are
cultured under flow conditions to produce a physiological shear
stress (.about.0.02 dyne/cm.sup.2). Kidney-specific microvascular
endothelial cells can be cultured on another side of the porous
membrane to form a Microvascular Channel as described above. Using
such kidney chips, the in vivo nephrotoxicity observed with an
active agent, e.g., cisplatin and its inhibition by cimetidine, can
be recapitulated and clinically relevant endpoints or biomarkers,
such as KIM-1, can be measured.
[0142] In some embodiments, the kidney chips described herein can
comprise components of the device, or can be the device described
in U.S. Provisional Application No. 61/449,925, the content of
which is incorporated herein by reference in its entirety.
[0143] Skin Chips:
[0144] Without limitations, in some embodiments, the skin chips can
be developed and/or modified, e.g., from the basic Lung Chip
multichannel design described herein (e.g., as shown in FIG. 2) as
well as in Huh D. et al., 2010 and/or in the International
Application No. WO 2010/009307, the contents of which are
incorporated herein by reference in their entireties. In some
embodiments, human foreskin fibroblasts can be plated in a collagen
gel on one side of the membrane, and human dermal endothelial cells
on the other side of the membrane; human keratinocytes from
foreskin can then be plated on top of the collagen gel layer.
[0145] In some embodiments, the cells seeded in the skin chips can
be further induced to differentiate into a stratified epithelium
with basal, spinous and cornified layers, e.g., by passing air
through the "Interstitial Channel" in a similar fashion as operated
in the lung chip, and creating an air-liquid interface.
[0146] Brain Chips:
[0147] Without limitations, in some embodiments, the brain chips
can be developed and/or modified, e.g., from the basic Lung Chip
multichannel design described herein (e.g., as shown in FIG. 2) as
well as in Huh D. et al., 2010 and/or in the International
Application No. WO 2010/009307, the contents of which are
incorporated herein by reference in their entireties. For example,
a brain chip can be constructed by first creating a Blood-Brain
Barrier (BBB) construct in which astrocytes (e.g., human
astrocytes) are cultured on one side of a porous ECM-coated
membrane and endothelial cells (e.g., human endothelial cells) on
the other side of the porous membrane. In some embodiments,
electrodes (e.g., platinum electrodes) are embedded inside the
channels, e.g., as shown in the bottom panel of FIG. 12. The
inventors have demonstrated generation of an effective permeability
barrier, as measured by transepithelial resistance using this
approach (FIG. 12).
[0148] In alternative embodiments, the brain chips can be developed
and/or modified, e.g., based on the design of any embodiments of
Heart Chips (e.g., as shown FIG. 8) described herein as well as in
Grosberg A. et al. 2011 and in the International Application Nos.
WO 2008/045506, WO 2010/011407 and WO2010/042856, the contents of
which are incorporated herein by reference in their entireties.
Such brain chips can be constructed, e.g., by placing the BBB
construct (as described above) in at least one set of channels and
then linking the outflow of its Interstitial Channel to a second
electrophysiological chamber (as described in the section of Heart
Chips) where human brain neuronal networks can be cultured to
measure effects on nerve cell toxicity and electrical signaling.
Without limitations, the brain chips described herein can be used
for various applications, e.g., discovery of methods to deliver an
active agent across a BBB, or as a Traumatic Brain Injury (TBI)
model to develop new therapies.
[0149] Skeletal Muscle Chips:
[0150] Without limitations, in some embodiments, the skeletal
muscle chips can be developed and/or modified, e.g., based on the
design of any embodiments of Heart Chips (e.g., as shown FIG. 8)
described herein as well as in Grosberg A. et al. 2011 and in the
International Application Nos. WO 2008/045506, WO 2010/011407 and
WO2010/042856, the contents of which are incorporated herein by
reference in their entireties.
[0151] Skeletal Muscle Chips are described, for example, in U.S.
Provisional Patent Application Ser. No. 61/569,028, filed on Dec.
9, 2011, U.S. Provisional Patent Application Ser. No. 61/697,121,
filed on Sep. 5, 2012, and PCT patent application titled "Muscle
Chips and Methods of Use Thereof," filed on Dec. 10, 2012 and which
claims priority to the U.S. provisional application No. 61/569,028,
filed on Dec. 9, 2011, U.S. Provisional Patent Application Ser. No.
61/697,121, the entire contents of all of which are incorporated
herein by reference in their entireties.
[0152] In some embodiments, the skeletal muscle chip can be a heart
chip adapted to culture mature skeletal muscle myoblasts or
myotubes (e.g., human skeletal muscle myoblasts or myotubes),
optionally with neurons, on 2D substrates (e.g., PDMS substrates)
or micromolded alginate substrates. These tissue constructs can
then be integrated into both a "muscular thin film" (MTF) chamber
for contractility measurements and a low volume
electrophysiological readout chamber, similar to the operation of
the heart chips described herein.
[0153] In some embodiments, the skeletal muscle chip can further
comprise additional cell types, e.g., adipocytes (e.g., human
adipocytes) can be added to the skeletal muscle chip as a
heterogeneous co-culture cell layer, or be seeded on a separate
side of the membrane to recapitulate two tissue interfaces, e.g.,
to create disease models within the device. For example, in one
embodiment, the adipocyte can be cultured on opposite side of
porous membrane that myoblast/neurons are cultured. The adipocytes
can be healthy or diseased, depending on the purpose of the
application. Healthy human adipocytes can be obtained from a
commercial source or isolated from a healthy tissue of a subject.
Alternatively, commercially-available healthy primary preadipocytes
(e.g., Lonza; PT-5022) can be differentiated into adipocytes, e.g.,
by using known differentiation medium. In some embodiments,
diabetic (Type I and II) human adipocytes can be used in the
skeletal muscle chip. For example, commercially-available primary
(from patients with diabetes) preadipocytes (e.g., Lonza;
PT-5023--Type I and PT-5024 Type II) can be differentiated into
adipocytes by using known differentiation medium.
[0154] To create an in vitro skeletal muscle system in a
microfluidic device, in some embodiments, aligned monolayers of
muscle cells (e.g., human myoblasts) can be cultured on
microcontact printed polymer substrates or membranes (e.g.,
microcontact printed PDMS substrates, microcontact printed
polyurethane membranes and/or micromolded alginate substrates). The
human myoblasts or satellite cells (healthy or diseased) can be
isolated from a tissue of a subject, and/or obtained from a
commercial source, e.g., (healthy; Lonza, XM13A1 and XM15B1).
[0155] In some embodiments, the skeletal muscle chip can comprise
at least one or more MTF chamber (FIGS. 13-14), e.g., at least two
or more MTF chambers. To create a MTF chamber, in one embodiment, a
laser engraving process can be utilized to cut PDMS thin films of
skeletal muscle cells (e.g., myoblasts and/or myotubes) cultured as
above; followed by microcontact-printing ECM proteins and enclosing
the assembly in a microfluidic chamber. To operate the MTF chamber,
muscle cells can be cultured on the thin films and the contractile
stresses can be assessed by optically monitoring the extent of
curvature of the muscular thin films. In some embodiments where the
muscle cells and adipose cells are co-cultured in the MTF chamber
(e.g., as shown in FIG. 14 (ii)), contractility readout from
skeletal muscle-adipocyte can be measured.
[0156] In some embodiments, the MTF chambers can be micromolded
with grooves (e.g., on an alginate substrate) to create 3D cues,
e.g., to help maintain mature contracting muscle in culture (FIGS.
15A-15C).
[0157] In some embodiments, the skeletal muscle chip can further
comprise an Electrophysiological (EPhys) chamber, the design and
operation of which can be similar to the ones employed in the heart
chips described herein, except that, e.g., the Ephys chamber is
cultured with skeletal muscle chips in the skeletal muscle chips.
By way of example only, to fabricate the EPhys chamber, anisotropic
muscle cell monolayer can be cultured in a low volume chamber.
Then, the muscle cells can be electrically field stimulated using
the electrodes on the top, and action potentials can be recorded
using the microelectrode array on the bottom. In some embodiments
where the muscle cells and adipose cells are co-culture in the
EPhys chamber, the EMG can be measured, e.g., as shown in FIG. 14
(i).
[0158] Prediction/Determination and Validation of Pharmacological
Effects:
[0159] The skeletal muscle chips described herein can be used to
determine pharmacological effects on skeletal muscle-specific cells
cultured therein. For example, the skeletal muscle-specific cells
can be exposed to an active agent by adding the active agent in a
culture medium flowing through the Interstitial Channels and/or
Microvascular Channels. In some embodiments where the skeletal
muscle chip comprises an Electrophysiological Chamber,
electrophysiological information can be collected within the chip,
which can be in turn modeled with a user-defined algorithm to
estimate an ECG. The ECG can then be correlated with reference data
(e.g., current in vitro and outputs, or control outputs, e.g.,
outputs of cells not exposed to the active agent). In one
embodiment, the radius of curvature of the contracting MTFs within
the chip can be modeled to estimate the stresses exerted by the
cells. The MTF contractility measurements can then be correlated
with reference data (e.g., current in vitro and clinical outputs,
or control outputs, e.g., outputs of cells not exposed to the
active agent).
[0160] For validation, in one embodiment, statin-induced skeletal
muscle myopathy can be replicated. Additionally or alternatively,
cerivastatin, which was voluntarily pulled from market in 2001 due
to side effects which included rhabdomyolysis and mypothay, can be
introduced into the skeletal muscle chips. Skeletal muscle damage
markers such as creatine kinase and slow and fast-twitch troponin I
(ssTnl, fsTnl) can also be measured (e.g., by in situ
immunostaining for the specific marker in the cells, and/or
collecting the cells to prepare cell lysates for quantitative
measurements of the marker level), in order to validate the ability
of the system to replicate injury induced by a known agent (e.g.,
statin-induced skeletal muscle myopathy). Additionally, since
muscle weakness is also a generalized complaint of patients
undergoing statin treatment, skeletal muscle contractility
measurements can also validate statin-induced in vitro muscle
weakness.
[0161] In some embodiments, the skeletal muscle chips can be used
as diseased models. For example, diabetic muscle microenvironment
can be created in some embodiments of the skeletal muscle chips
described herein, e.g., by co-culturing type I and type II
preadipocytes with the skeletal muscle cells. Type I diabetic mimic
chip can be used to evaluate insulin replacement therapies while
Type II diabetic mimic chip can be used to determine effectiveness
of metformin-induced metabolic benefits.
[0162] Further, an exemplary diabetic muscle environment (Type I
and II) can be created for pharmaceutical development testing. In
some embodiments of Type 2 diabetes model, adipocytes from type 2
diabetic patients (Lonza) and conditioned skeletal muscle can be
employed. Metformin, a first line drug used to treat type II
diabetes can then be introduced. Muscle contractility can be
measured to determine effectiveness of metformin-induced metabolic
benefits. In some embodiments of Type 1 diabetes model, Type 1
diabetic adipocytes along with conditioned muscle tissue in zero
insulin media can be exposed to commercially-used insulin
replacement therapies such as Detemir or Glargine. Improvements in
contractility can then be verified due to the increased
insulin.
[0163] Lung Airway Smooth Muscle Chips:
[0164] Without limitations, in some embodiments, the lung airway
smooth muscle chips can be developed and/or modified, e.g., based
on the design of any embodiments of Heart Chips (e.g., as shown
FIG. 8) described herein as well as in Grosberg A. et al. 2011 and
in the International Application Nos. WO 2008/045506, WO
2010/011407 and WO2010/042856, the contents of which are
incorporated herein by reference in their entireties.
[0165] In some embodiments, the lung airway smooth muscle chip can
be a hybrid bronchial smooth muscle and columnar epithelium organ
system, which can be used, e.g., to test bronchial spasms in
response to drugs and/or toxins. For example, as shown in FIG. 16B
(ii), this hybrid chip can be installed inside a two-chamber
microfluidic device that contains a top layer of epithelial cells
(e.g., human epithelial cells) adhered to a porous membrane and
exposed to air flow and a bottom layer containing flexible muscular
thin films, each comprising a monolayer (e.g., a confluent
monolayer) of bronchial smooth muscle situated on top of a thin
polymer substrate (e.g., a PDMS substrate), which are incubated
within a liquid medium. The porous membrane separating the top and
bottom layers of the chamber can act as a barrier between the two
cell types and allows for chemical exchange.
[0166] The material for the membrane that separates airway smooth
muscle from the epithelium within the hybrid organ system (e.g.,
lung airway smooth muscle chips) can be selected for at least one
of the following properties, but not limited to: the material is
(i) biocompatible, (ii) complies with IS 10993-5 (in vitro
cytotoxicity tests for medical devices), (iii) has low absorption
of hydrophobic dye/drug and other chemical compounds, (iv) is cell
adhesive, (v) is optically clear, is highly flexible, moldable,
bondable, (vi) has low autofluorescence, (vii) does not swell in
water, or (viii) has any combinations of the aforementioned
properties. In one embodiment, the membrane material can include
Clear flex 50 polyurethane.
[0167] In some embodiments, fabrication of an airway chip can
involve micro-contact printing to fabricate engineered smooth
muscle tissue and/or (e.g., human) columnar epithelium with
well-defined cellular organization and tissue geometry. Bronchial
smooth muscle cells (e.g., healthy or diseased cells) can be
obtained isolated from a tissue of a subject (e.g., a human),
and/or obtained from a commercial source (e.g., healthy: Lonza,
CC2576; diseased (COPD); Lonza, 00195274; diseased (asthmatic):
Lonza, 00194850).
[0168] Human primary bronchial epithelial cells (e.g., healthy or
diseased cells) can be isolated from a tissue or obtained from a
commercial source (e.g., healthy: ATCC; PCS-300-010, PCS-310-010,
Lonza: CC-2540, CC-2547; diseased: Lonza; 00195275, 00194911)
[0169] In some embodiments, a lung airway smooth muscle chip
("airway chip") can comprise a MTF chamber. To create a MTF
chamber, in one embodiment, a laser engraving process can be
utilized to cut PDMS thin films of muscle cells, e.g., bronchial
smooth muscle cells (the thin films can be produced in a similar
fashion as thin films of cardiomyocytes described above); followed
by microcontact-printing ECM proteins and enclosing the assembly in
a microfluidic chamber. To operate the MTF chamber, the muscle
cells, e.g., bronchial smooth muscle cells, can be cultured on the
thin films and the contractile stresses can be assessed by
optically monitoring, e.g., the extent of curvature of the muscular
thin films. In some embodiments, approximately 0.25 million muscle
cells or more can be seeded in each MTF chamber. An alternative
strategy for measuring muscle contraction is to grow muscle cells
on a wrinkling substrate, e.g., a PDMS membrane, such that as the
muscle cells contract, the substrate deforms. In this embodiment,
the wrinkling substrate can further comprise a plurality of holes
(e.g., a swiss cheese-like PDMS membrane) to facilitate optical
detection of the substrate deformation due to muscle cell
contraction. For example, the holes within the substrate can remain
as a circle when the muscle cells are relaxed, but the circle
becomes deformed, e.g., becoming an oval, or an ellipse, due to
muscle cell contraction, e.g., as shown in FIGS. 18A-18B.
[0170] In some embodiments, a two-chamber system can also be
engineered for dual outputs (e.g., as shown in FIGS. 17A-17B). For
example, normal bronchial epithelial cells can be cultured in one
chamber while diseased bronchial epithelial cells (e.g., with
asthma phenotype) can be cultured in another chamber, e.g., for
comparison of cell behavior, and/or cell response to an active
agent. In some embodiments, the cells can be chemically induced to
become diseased cells inside a microfluidic device, e.g., by
flowing an inducing agent through a separate channel to the
cells.
[0171] In some embodiments, epithelial cells can be cultured on the
chip membrane. In some embodiments, the epithelial cells can remain
viable for at least about 3 weeks or longer (e.g., at least about 4
weeks, at least about 5 weeks, at least about 6 weeks or longer)
inside the organ chip. For example, the epithelial cells can be
cultured on, e.g., 20.times.4 lined substrates, and the structural
and functional response of cells can be characterized by
quantifying cytoskeletal alignment and/or effects on gene
expression and/or protein translation.
[0172] In some embodiments, smooth muscles cells can be cultured on
a substrate (e.g., a deformable substrate). In some embodiments,
the smooth muscle cells can remain viable for at least 3 weeks or
longer (e.g., at least about 4 weeks, at least about 5 weeks, at
least about 6 weeks or longer) inside the airway chip. For example,
the smooth muscle cells can be cultured on, e.g., 20.times.4 lined
substrates, and the structural and functional response of cells
(e.g., cells on a deformable substrate exhibiting morphological
changes, e.g., contraction of such PDMS muscular thin film
substrate as in FIGS. 16A-16B and FIG. 17A-17B and/or deformation
of perforated holes in a membrane as in FIGS. 18A-18B), can be
characterized by quantifying cytoskeletal alignment and/or effects
on gene expression and/or protein translation.
[0173] To characterize cellular response to mechanical stimuli, the
bronchial smooth muscle tissue can be monitored for contraction in
response to an active agent, e.g., drug or toxic agents (e.g.,
without limitations, IL-13, acetylcholine). The contractility can
be measured for grading the response of the different tissue types
to the drugs.
[0174] In some embodiments, the lung airway smooth muscle chips can
comprise other "helper" cells. To characterize the cellular
response to different demographical conditions, e.g., addition of
"helper" cells, cells can be co-cultured conditions with other
"helper" cell types and/or different cell densities and their
effects on gene expression and protein translation can be evaluated
using any known methods in the art.
[0175] Prediction/Determination and Validation of Pharmacological
Effects:
[0176] The airway chips described herein can be used to determine
pharmacological effects on airway-specific cells cultured therein.
For example, the airway-specific cells can be exposed to an active
agent by adding the active agent in a culture medium flowing
through the channels. In one embodiment, the radius of curvature of
the contracting MTFs within the chip can be modeled to estimate the
stresses exerted by the cells. In another embodiment, the
eccentricity of the holes within a deformable membrane can be
evaluated to determine the state of the cellular contraction (e.g.,
relaxation vs. contraction state). The contractility measurements
(e.g., from contraction of MTFs) can then be correlated with
reference data (e.g., current in vitro and clinical outputs, or
control outputs, e.g., outputs of cells not exposed to the active
agent).
[0177] Local airway smooth muscle construct can be characterized
for any pharmacological mediators of interest, e.g., but not
limited to, Fexofenadine, Denufosol, Terfenadine, Isoproterenol,
Amiodarone, and Xigris. Characterization of airway smooth muscle
absorption can include, e.g., but not limited to, establishment of
absorptive rate constant (k.sub.a); establishment of
bioavailability (F): Determined by F=AUC.sub.INH/AUC.sub.IV and
determination of onset of action.
[0178] The in vitro microphysiological system (e.g., including two
or more organ chips) can also be characterized for any
pharmacological mediators of interest, e.g., but not limited to,
Fexofenadine, Denufosol, Terfenadine, Isoproterenol, Amiodarone,
and Xigris. Exemplary characterization include, without
limitations, establishment of duration of therapeutic effect;
establishment of apparent volume of distribution (V.sub.d), via
microfluidic channels within the system; establishment of Tmax,
time to maximal media concentration, per given dose, inhaled;
establishment of Cmax, maximal media concentration, per given dose,
inhaled; establishment of terminal half-life, per given dose,
inhaled; and/or establishment of efficacy of biotransformation as
result of first bypass effect at liver chip.
[0179] Performance of airway smooth muscle, local and systemic,
within an organ chip or an integrated system, can be corrected to
correspond to established pharmacokinetic and pharmacodynamics
values for various categories of drugs and toxins.
[0180] Disease Models:
[0181] In some embodiments, the lung airway smooth muscle chip can
be used as a disease model. The asthmatic phenotype can present as
exaggerated bronchial muscle spasms, which can be reflected by
changes in contractility. As a control, the effect of substances
with known toxicity in humans (e.g., local anesthetics such as
procaine, chloroprocaine, and tetracaine) can be introduced to
induce bronchial smooth muscle spasms and provide a calibration to
existing patient response from the clinic [Ref. 14].
[0182] A disease model can be developed, e.g., by integration of
diseased cells or by chemical stimulation of a diseased phenotype
(e.g., asthmatic phenotype can be induced, e.g., by introducing
IL-13 to the culture medium [Ref. 11]), and/or by alteration of
temperature and/or media composition [Ref. 12]. Optionally,
mechanical strain, electrophysiology, and/or markers for cell
injury between asthmatic phenotypes generated by integration of
cells from diseased tissues and those induced by allergens or
toxins can be compared and contrasted, e.g., to determine which is
a better representation of a diseased model.
[0183] By way of example only, to integrate the asthmatic muscle
phenotype into the microfluidic device, the asthmatic or chronic
obstructive pulmonary disease (COPD) human primary smooth muscle
cells (from either commercially available cell lines or samples
from asthmatic patients) can be cultured within one of the chambers
in the airway chip, e.g., on a deformable substrate, using
micro-contact printing to align cells into anisotropic tissues,
e.g., as shown in FIG. 19A. Contraction in smooth muscle cell layer
can be monitored optically as described above. In the same channel,
the micronenvironment conditions, e.g., the levels of oxygen within
the medium, cellular ATP, apoptosis, and/or intracellular calcium
can be also monitored.
[0184] Alternatively, the asthmatic phenotype can be induced by
introduction of allergens/drug compounds that drive bronchial
spasms associated with asthmatic attacks. For example, toxic agents
(e.g., acetylcholine, IL-13, Foradil, Serevent, Advair, Symbicort)
can be perfused into the culture medium containing the muscle cell
monolayer (FIG. 19B). Alteration in smooth muscle contraction in
response to allergens or toxic agents can be monitored optically.
In the same channel, the levels of oxygen within the medium,
cellular ATP, apoptosis, and/or intracellular calcium can be also
monitored.
[0185] In some embodiments where the upper channel containing an
epithelium (e.g., in a hybrid system as shown in FIG. 16 (ii)), the
levels of mucus granules and/or spherules and/or presence of
inflammation factors (e.g., but not limited to, IL-3, IL-4, TL-5,
IL-13) can be monitored.
[0186] The asthmatic phenotype can be induced by introduction of
allergens/drug compounds through the air, allergens (e.g. oil fly
ash, dust mites, smoke) can be perfused into the aerosol routes
within the upper channel of the microfluidic device containing the
epithelium (see, e.g., FIG. 16B(ii)). Alterations in smooth muscle
contraction in response to allergens or toxic agents can be
monitored optically. In the same channel, microenvironment
conditions, e.g., but not limited to, the levels of oxygen within
the medium, cellular ATP, apoptosis, and/or intracellular calcium
can be monitored. In the upper channel containing the epithelium,
the levels of mucus granules and/or spherules and/or presence of
inflammation factors (e.g., but not limited to, IL-3, IL-4, IL-5,
IL-13) can be monitored.
[0187] A diseased epithelial cell monolayer can be also integrated
within the asthmatic microfluidic device, e.g., a co-culture of the
asthmatic or COPD human primary smooth muscle cells (from either
commercially available cell lines or samples from asthmatic
patients) with the diseased human primary bronchial epithelial
cells. By way of example only, the diseased epithelial cell
monolayer can be cultured on the membrane (e.g., polyurethane
membrane) and the diseased smooth muscle cells on the deformable
substrate, using micro-contact printing to align cells into
anisotropic tissue in a hybrid system described herein (e.g., as
shown in FIG. 16B (ii)). Contraction in smooth muscle cell layer
can be monitored optically. In the same channel, microenvironment
conditions, e.g., but not limited to, the levels of oxygen (e.g.,
reduction in oxygen levels) within the medium, cellular ATP,
apoptosis, and/or intracellular calcium can be monitored. In the
upper channel containing the epithelium, the levels of mucus
granules and/or spherules and/or presence of inflammation factors
(e.g., but not limited to, IL-3, IL-4, IL-5, IL-13) can be
monitored. Further, the hyperplasia and hypertrophy of the columnar
epithelial cells (hallmarks of obstructive pulmonary disease) can
be assessed optically, e.g., by microscopy.
[0188] Asthma is a prevalent disease affecting .about.8% percent of
the population, and it is in part due to maladaptive remodeling of
bronchial smooth muscle. Airborne toxins and air pollution can
affect the bronchial smooth muscle, but they are difficult to
detect and to predict their effects on the tissue health (and
reflect a large scale problem in developing countries). There are
currently no commercially available instruments to test
cytotoxicity and allergic responses and associated inflammation in
vitro, despite the existence of human bronchial smooth muscle cell
lines. Beyond the difference in species responses to toxic or
therapeutic agents, animal models do not necessarily provide human
relevance, predictability, and lower failure rates in the drug
pipe-line.
[0189] Bronchial smooth muscle spasms can be a response to
allergens and/or toxins. This response can be amplified if the
muscle undergoes a maladaptive remodeling prior to the introduction
of the toxic agent (such as in asthmatic patients). Therefore, an
in vitro organ chip system (e.g., a hybrid chip of bronchial smooth
muscle tissue and epithelial cell monolayer as described herein) to
test for bronchial smooth muscle spasms can provide a tool for
various applications, including, but not limited to: testing drugs
for curing or exacerbating asthma; testing toxins (including
air-borne toxins) in the field; predicting the allergic response of
different patient populations. Further, the hybrid chips can be
combined with a microfluidic device containing bronchial epithelial
and/or pulmonary mast cells, which are important to exacerbation
and inflammation response. In other embodiments, the hybrid chips
described herein can comprise bronchial epithelial and/or pulmonary
mast cells within the same device. In some embodiments where
epithelial and mast cells are co-cultured, the epithelial and mast
cells can produce inflammation factors (e.g., but not limited to,
IL-3, IL-4, IL-5 IL-13) that would cause the spasms in the
bronchial smooth muscle, and the levels of these inflammation
factors can be measured to evaluate the degree of spasms.
[0190] An exemplary protocol of fabricating a lung smooth muscle
chip and using the same for evaluating cell response to a drug is
shown below: (Any modifications to the protocol within one of skill
in the art are also within the scope of the inventions.) [0191]
Create a chip with a desirable number of chambers, e.g., one or
more chambers (FIG. 16A(i)-(ii)); [0192] Control cell shape and
tissue alignment within the chambers to recapitulate the native
microenvironments. This can be achieved, e.g., by either patterning
of ECM, microgrooves, stretching of the membrane, or other similar
techniques as described herein; [0193] Seed cells and supply media
through micro-fluidic channels (FIG. 16A(i)). The bronchial smooth
muscle cells (e.g., human cells) can be obtained from commercially
available cell-lines [Ref. 10] and/or primary cells from a subject,
e.g., human healthy or asthmatic patients; [0194] The organ chip
can also allow for co-culture with other cell types: [0195]
Bronchial epithelial cells can be cultured within the same chamber
separated by a membrane (e.g., a transwell membrane), and exposed
to exacerbating pollutants, such as oil fly ash (available
commercially), dust mites, or smoke; [0196] The bronchial smooth
muscle cells can be combined with pulmonary mast cells either
upstream in the microfluidic device or co-cultured within a
transwell chamber; [0197] Induce an asthmatic phenotype chemically
(e.g., IL-13 during culture [Ref. 11]), by temperature control,
and/or alteration of media composition [Ref. 12] in some of the
chambers (FIG. 17A(i), chamber 2). Additionally or alternatively,
the asthmatic phenotype can be incorporated into the chip by
samples derived from asthmatic patients [0198] If desired, add a
therapeutic drug to the asthmatic chamber [0199] Introduce a drug
to induce a normal smooth muscle contractile response, and/or a
toxic agent to test for bronchial spasms (e.g., acetylcholine)
[0200] Contraction of the muscle can be detected using multiple
approaches, or any approaches known in the art. Approaches 2 and 3
(below) can be used to mimic the function of the bronchial muscle
where the cells close-off micro-holes the same way as they
close-off the bronchia (Without construed to be limiting, various
approaches described below to measure cell contraction can also be
integrated into any organ chips described herein): [0201] 1. By
making the chamber substrates out of PDMS muscular thin films, it
can be used to optically track the bending of each film as a
measure of contractility (e.g., FIGS. 16A-16B) [0202] 2.
Alternatively, a substrate can be created with micro-holes, which
are small enough to be impermeable to the cells. These holes can
provide the material with greater flexibility, and their shape can
be used as an indicator of the amount of contraction by reading out
their shape based on the transmitted light (FIGS. 18A-18B); [0203]
3. Rows of oval micro-holes can be created with varying
eccentricity, such that conduction is possible through the wires in
the material if the holes are completely closed. The row of
micro-holes with minimal eccentricity that still conducts can be
used to provide the readout of the degree of contraction of the
tissue; and/or [0204] 4. Materials that are capable of varying
color with different degrees of stress can also be used; [0205] The
output readout of the contractility and proliferation of the
bronchial smooth muscle can then be related to lung capacity curves
(rate of inhale/exhale) through the modeling: [0206] 1. Average
material properties of the endothelial layer can be estimated from
histological data; [0207] 2. Stiffness of the muscle layer can be
calculated from the in vitro experimental data (from chip); and/or
[0208] 3. These can be used to calculate the diameter of the
bronchia tube, which can then be used to calculate the
airflow/resistance--clinical outputs
[0209] In some embodiments, the chip's function can be calibrated
by introducing a substance with known allergenicity, such as
procaine, chloroprocaine, and tetracaine, which have been used in
humans as local anesthetics and have been shown to be a plausible
allergen that can induce a bronchial smooth muscle spasm [4]. This
calibration can also be compared to known patient responses, which
can provide the necessary data to predict human response based on
the data retrieved from the chip.
[0210] For a more recent drug, Raplon (Rapacuronium, Organon) is an
anesthetic that was approved in the USA in 1999 and withdrawn by
the FDA two years later due to a high risk of fatal bronchial
muscle spasm [Ref. 14]. Additionally, drugs such as Novartis AG's
Foradil, GlaxoSmithKline's Serevent and Advair, and AstraZeneca's
Symbicort, while approved to treat asthma, can themselves cause
asthma attacks [Ref. 13]. The chip design described herein can
allow for in vitro cultures to run for long periods of time to test
such effects, prior to clinical trials.
[0211] In some embodiments, the lung airway smooth muscle chips can
be connected to at least one lung chip to function as an integral
organ chip. In some embodiments, the design of the lung airway
smooth muscle can be incorporated into the lung chip design.
[0212] In some embodiments, the airway chip can be integrated into
the system with other organ chips, which can then be used to
evaluate drugs that are effective at treating bronchial smooth
muscle tissues, but might be toxic to other organ systems (e.g.,
Ventolin). Alternatively, such integrated system can also allow for
testing of drugs commonly used to affect the function of other
organs (e.g., beta blockers that reduce cardiac contractility), but
can adversely affect airway resistance in asthmatics.
[0213] Drugs can be administered via both intravenous (e.g., via
culture medium) and aerosol routes, exhibiting the versatility of
the chip design.
[0214] The airway muscle chips, alone or in combinations with other
organ chips, can be used in various applications. Non-limiting
examples of applications are shown below: [0215] Provide an in
vitro platform for bronchial smooth muscle spasm testing; [0216]
Provide a platform to test the contractility of human bronchial
smooth muscle tissue; [0217] Primary cells isolated from different
population of patients can be used with this platform to study the
effectiveness of drugs for different subsets of the population;
[0218] Provide key endpoints--translating chip outputs to clinical
diagnostic tools: [0219] The chip readout of the contractility and
proliferation of the bronchial smooth muscle can be related to lung
capacity curves (rate of inhale/exhale) and compared to the output
of pulmonary function tests in the clinic; [0220] The secretion of
mucus granules and spherules in the epithelium can be monitored
optically as a means to test for mucous secretion that is a
hallmark of bronchitis and other obstructive pulmonary diseases;
and/or [0221] Patients with advanced chronic obstructive pulmonary
disease like chronic bronchitis often exhibit bluish tinted skin,
resulting from hypoxia and fluid retention. Thus, the media can be
tested for low oxygen levels and arterial blood gas (measuring
ability to oxygenate blood at alveoli) "downstream" of obstructed
airways.
[0222] Testis Chips:
[0223] Without limitations, in some embodiments, the Testis Chip
can be developed and/or modified, e.g., from the basic Lung Chip
multichannel design described herein (e.g., as shown in FIG. 2) as
well as in Huh D. et al., 2010 and/or in the International
Application No. WO 2010/009307, the contents of which are
incorporated herein by reference in their entireties. In some
embodiments, the testis chips can employ (e.g., human) Sertoli and
Leydig cells being cultured on one side of the porous ECM-coated
membrane and endothelium on the other side of the coated membrane.
This Testis chip design can maintain enhanced differentiated
testicular functions when the two parenchymal cell types are
combined in this manner (Ref. 4).
[0224] Bone Marrow Chips:
[0225] To construct a Bone Marrow chip, fully functional bone
containing a central marrow can be formed in vivo first by
implanting demineralized bone powder and BMPs 2/4 subcutaneously
above a muscle layer within a polymer mold (e.g., PDMS mold and
culturing it for about 4-8 weeks in vivo. Additional details of the
bone marrow chips and methods of making the same can be found in
the International Appl. No. PCT/US12/40188, the content of which is
incorporated herein by reference in its entirety. The bones that
form in these implanted devices can take the shape (e.g.,
cylindrical shape) of the flexible mold, and contain a fully
developed bone marrow with normal morphology and cellular
composition (hematopoietic stem cells, progenitor cells, various
differentiated blood cell types), when compared to normal mouse
bone marrow versus peripheral blood (FIG. 20). The formed marrow
can be maintained by placing the formed implant within microfluidic
channels, as evidenced by cells isolated from this marrow after 4
days in culture being able to regenerate a functional marrow and
reconstitute whole blood formation in gamma-irradiated mice.
[0226] Alternatively, simultaneously reconstituting the mouse's
injured marrow and forming new marrow in the microfluidic implants
can be carried out by irradiating immuno-compromised mice,
implanting the demineralized bone powder with BMPs subcutaneously,
and then injecting human bone marrow. Once removed and maintained
in microfluidic systems, the human marrow can then be used to
generate all types of blood cells, which can circulate throughout
the entire linked organ chip circuit, e.g., an in vitro
microphysiological system, e.g., for studies on inflammation and
its relation to drug toxicity.
[0227] Without limitations, additional organ chips corresponding to
other organs, e.g., spleen chips for filtration of fluid, e.g.,
blood, as described into the International Appl. No. WO
2012/135834, the content of which is incorporated here by reference
in its entirety, can also be integrated into an in vitro
microphysiological system described herein.
Exemplary Applications of Organ Chips and/or In Vitro
Microphysiological Systems Described Herein
[0228] At least one or more organ chips can be used for any
applications that involve cells. In some embodiments, the organ
chips and/or in vitro microphysiological systems can be used as
cell culture devices. Compared to 2-D tissue culture flasks, the
organ chips and/or in vitro microphysiological systems described
herein can provide organ-specific cells a more physiological
condition for their growth, and/or maintenance of their
differentiated states. For example, lung cells in vivo are
generally exposed to a mechanical stimulation, e.g., during
breathing. To mimic the breathing action in vitro, organ chips such
as lung chips can be used to culture lungs cells as described
above. In some embodiments, the cells can be cultured and remain
viable (e.g., capable of proliferation) for at least about 3 weeks,
at least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 9 weeks, at least about 12 weeks or longer
inside the organ chips described herein.
[0229] In some embodiments, the organ chips and/or in vitro
microphysiological systems can be used for drug screening, PD/PK
studies and/or toxicity assays. For example, an active agent can be
delivered to target cells cultured in organ chips, e.g., by
introducing the active agent into the organ chips as an aerosol
and/or as a mixture with the culture medium flowing through the
Interstitial Channels and/or Microvascular Channels of the organs
chips described herein. Examples of active agents to be delivered
to target cells can include, but are not limited to, cells
including, e.g., but not limited to, bacteria and/or virus,
proteins, peptides, antigens, antibodies or portions thereof,
enzymes, nucleic acids, siRNA, shRNA, aptamers, small molecules,
antibiotics, drugs or therapeutic agents, molecular toxins,
nanomaterials or particulates, aerosols, environmental contaminants
or pollutants (e.g., but not limited to, microorganisms,
organic/inorganic contaminants present in food and/or water, and/or
air pollutants), and any combinations thereof. Exemplary
characterization that can be performed with the organ chips and/or
in vitro microphysiological systems described herein can include,
without limitations, establishment of duration of therapeutic
effect; establishment of apparent volume of distribution (Vd), via
microfluidic channels within the system; establishment of Tmax,
time to maximal media concentration, per given dose; establishment
of Cmax, maximal media concentration, per given dose; establishment
of terminal half-life, per given dose; and/or establishment of
efficacy of biotransformation as result of first bypass effect at
the organ chip.
[0230] Examples of drugs can include, but are limited to,
pharmacologically active drugs and genetically active molecules.
Compounds of interest include chemotherapeutic agents,
anti-inflammatory agents, hormones or hormone antagonists, ion
channel modifiers, and neuroactive agents. Exemplary of
pharmaceutical agents suitable for this invention are those
described in "The Pharmacological Basis of Therapeutics," Goodman
and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition,
under the sections: Drugs Acting at Synaptic and Neuroeffector
Junctional Sites; Drugs Acting on the Central Nervous System;
Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions;
Drugs Affecting Renal Function and Electrolyte Metabolism;
Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function;
Drugs Affecting Uterine Motility; Chemotherapy of Parasitic
Infections; Chemotherapy of Microbial Diseases; Chemotherapy of
Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting
on Blood-Forming Organs; Hormones and Hormone Antagonists;
Vitamins, Dermatology; and Toxicology, all incorporated herein by
reference. Also included are toxins, and biological and chemical
warfare agents, for example see Somani, S. M. (Ed.), "Chemical
Warfare Agents," Academic Press, New York, 1992).
[0231] Additional candidate compounds or drugs can be obtained from
a wide variety of sources including libraries of synthetic or
natural compounds. For example, numerous means are available for
random and directed synthesis of a wide variety of organic
compounds and biomolecules, including expression of randomized
oligonucleotides and oligopeptides. Alternatively, libraries of
natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
naturally or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means, and may be used to produce combinatorial
libraries. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0232] By way of example only, the cells within the organ chips
and/or in vitro microphysiological systems can be exposed to a drug
by flowing a fluid containing the drug through a channel of an
organ chip or an integrated system of different organ chips, such
that the fluid is in contact with the cells. In some embodiments,
the cells within the organ chips and/or in vitro microphysiological
systems can be exposed to an aerosol by flowing a gaseous fluid
containing the aerosolized microdroplets of a drug through a
channel of an organ chip or an integrated system of different organ
chips such that the gaseous fluid is in contact with the cells.
Cell response in one or more organ chips can then be measured after
or monitored over a period of time. Examples of cell response can
include, but are not limited to, viability, proliferation,
respiration, metabolism, movement of the cells (e.g., migration,
contractile motions), differential expression of biomarkers, and/or
production and/or release of certain molecules by the cells. Any
methods known in the art can be utilized for detecting or measuring
a cell response, e.g., immunostaining, microscopy, immunoassays,
PCR, and/or ECG measurements.
[0233] In some embodiments where the cells are collected from a
subject, a treatment regimen (e.g., a therapeutic agent and/or
dosage that works best, among others, for the subject) can be
selected and/or optimized by culturing the subject-specific cells
in the organ chips and/or in vitro microphysiological systems
described herein, exposing the cells to different therapeutic
agents and/or dosages, and monitoring the cellular response to
various combinations.
[0234] In some embodiments, the cells can be observed by microscopy
for morphological changes. In some embodiments, the contractile
motion of the cells can be measured by ECG measurements. In some
embodiments, the cells can be stained for a target protein, e.g., a
biomarker, and then observed under a microscope. In some
embodiments, the cells can be collected from the organ chips for
further analysis, such as RNA, DNA and/or protein analysis. In some
embodiments, the culture medium conditioned by the cells can be
collected for further analysis, such as RNA, DNA and/or protein
analysis. One of skill in the art can readily perform various
assays for detecting or measuring different kinds of cell
responses.
[0235] In some embodiments, the organ chips (e.g., gut chips)
and/or in vitro microphysiological systems described herein can be
used for studying the role of gut flora (e.g., microorganisms that
live in the digestive tracts of animals) and other bacteria within
a body of an animal that can have a symbiotic relationship with the
host. Various factors other than infections, such as aging,
geographical transplant, changes in diet, and/or various
therapeutic regimens such as antibiotics can alter the gut flora
demographics and the physiology of the host. See, e.g., Maynard C L
et al. "Reciprocal interactions of the intestinal microbiota and
immune system." Nature. 2012 Sep. 13; 489(7415):231-41; Tremaroli
V. and Backhed F. "Functional interactions between the gut
microbiota and host metabolism." Nature. 2012 Sep. 13;
489(7415):242-9; Lozupone C A et al. "Diversity, stability and
resilience of the human gut microbiota" Nature. 2012 Sep. 13;
489(7415):220-30; and Ottman N et al. "The function of our
microbiota: who is out there and what do they do?" Front Cell
Infect Microbiol. 2012; 2:104. Epub 2012 Aug. 9, for information on
gut microbiome and human health/disease. For example, C. difficile
is a serious cause of antibiotic-associated diarrhea (AAD) and can
lead to pseudomembranous colitis, a severe inflammation of the
colon, often resulting from eradication of the normal gut flora by
antibiotics. Accordingly, in some embodiments, "cassettes" of gut
bacteria colonies can be co-cultured with gut cells and/or
intestine cells in appropriate organ chips, e.g., gut chips, for
example, to model gut flora in a host, and/or to study the effects
of different factors on the gut flora demographics and/or
physiology of the host cells. In some embodiments, these gut chips
can be connected to other organ chips to form in vitro
microphysiological systems that can be desirable when considering
the mind body axis and the coupling of the enteric and central
nervous system. These systems can be also used to study, e.g., but
not limited to, digestion, and mental illness.
[0236] In some embodiments, methods to study microbial growth,
adhesion to host-related surfaces and/or the host-microbiota
interactions, e.g., as described in the U.S. Pat. App. No. US
2012/0058551, the content of which is incorporated herein by
reference, can be integrated or utilized together with the organ
chips and/or in vitro microphysiological system described herein to
study the role of gut flora within a body of an animal.
[0237] In some embodiments, the organ chips and/or in vitro
microphysiological systems described herein can be used to study
and/or model parasitic infections, for example, but not limited to,
co-culture of intestinal worms, rabies, toxoplasma, and any
combinations thereof.
Kits
[0238] Another aspect provided herein relates to kits comprising
one or a plurality of organ chips, for example, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or more organ chips. In some embodiments, the organ
chips in the kit can be all the same, i.e., corresponding to the
same organ. In some embodiments, at least some of the organ chips
in the kit can represent a different organ. In some embodiments,
the organ chips in the kit can be pre-assembled together, or
fluidically connected together by a user, to form an integrated
microphysiological system. For example, a kit directed to a
circulatory system can comprise at least one heart chip and at
least one bone-marrow chip. Another kit directed to a
gastrointestinal system can comprise at least one liver chip and
one gut chip. Depending on the microphysiological system of
interest, the kits can comprise a plurality of the organ chips that
are involved in the microphysiological system.
[0239] In some embodiments, each organ chips can be individually
packaged, e.g., for sterility. In some embodiments, the kits can
further comprise an appropriate culture medium. In some
embodiments, the kit can further comprise at least one vial of
cells, e.g., vascular endothelial cells and/or organ-specific
parenchymal cells. In some embodiments, the kits can further
comprise an instruction manual, e.g., instructions on connecting
various organ chips together to form an integrated network, cell
culture methods, and approaches of measuring various cellular
responses.
Fabrications of the Organ Chips Described Herein
[0240] The methods used in fabrication of any embodiments of the
organ chip described herein can vary with the materials used, and
include soft lithography methods, microassembly, bulk
micromachining methods, surface micro-machining methods, standard
lithographic methods, wet etching, reactive ion etching, plasma
etching, stereolithography and laser chemical three-dimensional
writing methods, solid-object printing, machining, modular assembly
methods, replica molding methods, injection molding methods, hot
molding methods, laser ablation methods, combinations of methods,
and other methods known in the art. A variety of exemplary
fabrication methods are described in Fiorini and Chiu, 2005,
"Disposable microfluidic devices: fabrication, function, and
application" Biotechniques 38:429-446; Beebe et al., 2000,
"Microfluidic tectonics: a comprehensive construction platform for
microfluidic systems." Proc. Natl. Acad. Sci. USA 97:13488-13493;
Rossier et al., 2002, "Plasma etched polymer microelectrochemical
systems" Lab Chip 2:145-150; Becker et al., 2002, "Polymer
microfluidic devices" Talanta 56:267-287; Becker et al., 2000,
"Polymer microfabrication methods for microfluidic analytical
applications" Electrophoresis 21:12-26; U.S. Pat. No. 6,767,706 B2,
e.g., Section 6.8 "Microfabrication of a Silicon Device"; McDonald
et al., 2002, "Poly(dimethylsiloxane) as a material for fabricating
microfluidic devices" Accounts of Chemical Research 35: 491-499.
Piccin et al., 2007, "Polyurethane from biosource as a new material
for fabrication of microfluidic devices by rapid prototyping"
Journal of Chromatography A 1173: 151-158. Each of these references
is incorporated herein by reference.
[0241] In some embodiments, an organ chip described herein can be
formed by replica molding, for example, in which a replica
comprising at least biocompatible polymer (e.g., PDMS polymer)
conforms to the shape of a master or a mold and replicates the
features of the master or the mold. In some embodiments, the
replica can be further sealed to a surface to enclose at least one
channel.
[0242] In some embodiments, an organ chip described herein can be
formed by machining or micromachining. The term "micromachining" as
used herein can encompass bulk micromachining or surface
micromachining as recognized in the art. In one embodiment, bulk
micromachining defines microstructures such as channels by
selectively etching inside a substrate or a body.
[0243] In some embodiments, an organ chip described herein can be
formed by solid-object printing. In some embodiments, the
solid-object printing can take a three-dimensional (3D)
computer-aided design file to make a series of cross-sectional
slices. Each slice can then be printed on top of one another to
create the 3D solid object.
[0244] Embodiments of the various aspects described herein can be
illustrated by the following numbered paragraphs. [0245] 1. An in
vitro microphysiological system comprising: [0246] a. at least two
different organ chips, wherein said at least different two organ
chips are selected from either one or both of the following: [0247]
(i) a first organ chip comprising: a body comprising a central
channel therein, and an least partially porous and at least
partially flexible first membrane positioned within the central
channel and along a plane, wherein the first membrane is configured
to separate the central channel to form two sub-channels, wherein
one side of the first membrane is seeded with vascular endothelial
cells, and the other side of the first membrane is seeded with at
least one type of organ-specific parenchymal cells; [0248] (ii) a
second organ chip comprising: a body comprising a first chamber
enclosing a plurality of muscular thin films adapted to measure
contraction of muscle cells, and a second chamber comprising a
layer of muscle cells on the bottom surface of the second chamber,
wherein the bottom surface is embedded with an array of
microelectrodes for recording of action potentials, and wherein the
top surface of the second chamber is placed with at least a pair of
electrodes for providing electric field stimulation to the muscle
cells; or [0249] (iii) a combination of the first organ chip and
the second organ chip; and [0250] b. at least one connecting means
between said at least two different organ chips. [0251] 2. The
system of paragraph 1, wherein the system comprises at least three
organ chips. [0252] 3. The system of paragraph 1 or 2, wherein the
connecting means comprises a tubing that fluidically connects an
outlet of one of the organ chips to an inlet of another organ chip.
[0253] 4. The system of any of paragraphs 1-3, wherein the first
organ chip is selected from the group consisting of a lung chip, a
liver chip, a gut chip, a kidney chip, a skin chip, a brain chip, a
testis chip, and any combinations thereof. [0254] 5. The system of
any of paragraphs 1-3, wherein the second organ chip is selected
from the group consisting of a heart chip, a skeletal muscle chip,
a lung airway smooth muscle chip, a brain chip, and any
combinations thereof. [0255] 6. The system of any of paragraphs
1-5, further comprising a bone marrow chip fluidically connected to
said at least two different organ chips. [0256] 7. The system of
any of paragraphs 1-6, further comprising a spleen chip fluidically
connected to said at least two different organ chips. [0257] 8. The
system of any of paragraphs 1-7, wherein the first organ chip
further comprises at least a channel wall positioned adjacent to
the two sub-channels, wherein the first membrane is mounted to the
channel wall; and an operating channel adjacent to the two
sub-channels on an opposing side of the channel wall, wherein a
pressure differential applied between the operating channel and the
two sub-channels causes the channel wall to flex in a desired
direction to expand or contract along the plane within the two
sub-channels. [0258] 9. The system of any of paragraphs 1-8,
wherein the second organ chip further comprises an at least
partially porous second membrane positioned within the first
chamber to form a top chamber and a bottom chamber, wherein the
bottom chamber comprises the plurality of muscular thin films on
its bottom surface, and wherein the surface of the second membrane
in contact with the top chamber is seeded with a layer of
epithelial cells. [0259] 10. The system of any of paragraphs 1-9,
wherein when the system comprises a circulatory system, said at
least two different organ chips comprise a heart chip and a bone
marrow chip. [0260] 11. The system of any of paragraphs 1-10,
wherein when the system comprises a respiratory system, said at
least two different organ chips comprise a lung chip and an airway
smooth muscle chip. [0261] 12. The system of any of paragraphs
1-11, wherein when the system comprises an excretory system, said
at least two different organ chips comprise a lung chip, a gut
chip, and a kidney chip. [0262] 13. The system of any of paragraphs
1-12, wherein when the system comprises a nervous system, said at
least two different organ chips comprise a brain chip and a chip
with neuronal networks. [0263] 14. The system of any of paragraphs
1-13, wherein when the system comprises a gastrointestinal system,
said at least two different organ chips comprise a liver chip, and
a gut chip. [0264] 15. The system of any of paragraphs 1-14,
wherein the system is adapted to determine at least one
pharmacokinetic and/or pharmacodynamics parameter of an active
agent. [0265] 16. The system of paragraph 15, wherein the active
agent is selected from the group consisting of cells, proteins,
peptides, antigens, antibodies or portions thereof, antibody-like
molecules, enzymes, nucleic acids, siRNA, shRNA, aptamers, small
molecules, antibiotics, therapeutic agents, molecular toxins,
nanomaterials, particulates, aerosols, environmental contaminants
or pollutants, and any combinations thereof. [0266] 17. The system
of any of paragraphs 1-16, wherein said at least two different
organ chips are connected in parallel. [0267] 18. The system of any
of paragraphs 1-17, wherein said at least two different organ chips
are connected in series. [0268] 19. The system of any of paragraphs
1-18, further comprising a housing enclosing said at least two
different organ chips. [0269] 20. The system of any of paragraphs
1-19, wherein said at least two different organ chips comprise at
least two said first organ chips. [0270] 21. The system of any of
paragraphs 1-20, wherein said at least two different organ chips
comprise at least two said second organ chips. [0271] 22. A kit
comprising: [0272] a. at least one in vitro microphysiological
system of any of paragraphs 1-21; and [0273] b. at least one agent.
[0274] 23. The kit of paragraph 22, wherein said at least one agent
comprises a culture medium, an agent for calibration and/or
validation of the system, or a combination thereof. [0275] 24. The
kit of any of paragraphs 22-23, further comprising at least one
vial of vascular endothelial cells. [0276] 25. The kit of any of
paragraphs 22-24, further comprising at least one vial of
organ-specific parenchymal cells. [0277] 26. The kit of any of
paragraphs 22-25, wherein the organ chips involved in the
microphysiological system are each individually packaged. [0278]
27. A method comprising: [0279] a. contacting either one or both of
the organ-specific parenchymal cells and the muscle cells cultured
in said at least two different organ chips of the in vitro
microphysiological system of any of paragraphs 1-21 with at least
one active agent for a period of time; [0280] b. measuring a
response of the cells cultured in said at least two different organ
chips of the in vitro microphysiological system to determine the
effect of the at least one active agent on at least two organs.
[0281] 28. The method of paragraph 27, wherein the cells are
contacted with said at least one active agent by flowing the active
agent through the channel or chamber where the cells are cultured.
[0282] 29. The method of paragraph 28, wherein the active agent is
added to a liquid flowing through the channel or the chamber.
[0283] 30. The method of paragraph 29, wherein the liquid comprises
cell culture medium, blood, or a combination thereof. [0284] 31.
The method of paragraph 28, wherein the active agent is in a form
of an aerosol flowing through the channel or the chamber. [0285]
32. The method of any of paragraphs 27-31, wherein the response of
the cells is selected from the group consisting of viability,
proliferation, respiration, metabolism, cell migration, cell
contractility, differential expression of cell biomarkers,
production and/or release of biomolecules, action potentials, and
any combinations thereof. [0286] 33. The method of any of
paragraphs 27-32, wherein the organ-specific parenchymal cells, the
muscle cells, or both are collected from a subject. [0287] 34. The
method of any of paragraphs 27-32, wherein the organ-specific
parenchymal cells, the muscle cells, or both are differentiated
from stems cells collected from a subject. [0288] 35. The method of
paragraph 33 or 34, further comprising comparing the responses of
the cells to that of control cells not contacted with said at least
one active agent. [0289] 36. The method of any of paragraphs 27-35,
wherein the active agent is selected from the group consisting of
cells, proteins, peptides, antigens, antibodies or portions
thereof, antibody-like molecules, enzymes, nucleic acids, siRNA,
shRNA, aptamers, small molecules, antibiotics, therapeutic agents,
molecular toxins, nanomaterials, particulates, aerosols,
environmental contaminants or pollutants, and any combinations
thereof. [0290] 37. The method of paragraph 36, wherein when said
at least active agent comprises a therapeutic agent, the method
further comprises selecting a treatment regimen comprising the
therapeutic agent. [0291] 38. The method of paragraph 37, further
comprising administering the therapeutic agent to the subject.
[0292] 39. The method of any of paragraphs 33-38, wherein the
subject is a human subject. [0293] 40. The method of any of
paragraphs 27-39, further comprising growing the cells for at least
about 3 weeks.
Some Selected Definitions
[0294] Unless stated otherwise, or implicit from context, the
following terms and phrases include the meanings provided below.
Unless explicitly stated otherwise, or apparent from context, the
terms and phrases below do not exclude the meaning that the term or
phrase has acquired in the art to which it pertains. The
definitions are provided to aid in describing particular
embodiments of the aspects described herein, and are not intended
to limit the claimed invention, because the scope of the invention
is limited only by the claims. Further, unless otherwise required
by context, singular terms shall include pluralities and plural
terms shall include the singular.
[0295] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0296] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of additional elements that do not materially affect
the basic and novel or functional characteristic(s) of that
embodiment of the invention.
[0297] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0298] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages may mean.+-.1%.
[0299] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. Thus for example, references to "the
method" includes one or more methods, and/or steps of the type
described herein and/or which will become apparent to those persons
skilled in the art upon reading this disclosure and so forth.
[0300] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
this disclosure, suitable methods and materials are described
below. The term "comprises" means "includes." The abbreviation,
"e.g." is derived from the Latin exempli gratia, and is used herein
to indicate a non-limiting example. Thus, the abbreviation "e.g."
is synonymous with the term "for example."
[0301] As used herein, a "subject" means a human or animal. Usually
the animal is a vertebrate such as a primate, rodent, domestic
animal or game animal. Primates include chimpanzees, cynomologous
monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents
include mice, rats, woodchucks, ferrets, rabbits and hamsters.
Domestic and game animals include cows, horses, pigs, deer, bison,
buffalo, feline species, e.g., domestic cat, canine species, e.g.,
dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and
fish, e.g., trout, catfish and salmon. In certain embodiments of
the aspects described herein, the subject is a mammal, e.g., a
primate, e.g., a human. A subject can be male or female.
Preferably, the subject is a mammal. The mammal can be a human,
non-human primate, mouse, rat, dog, cat, horse, or cow, but are not
limited to these examples. Mammals other than humans can be
advantageously used as subjects that represent animal models of a
disease or disorder.
[0302] As used herein, the term "fluid" refers to any flowable
material or medium, e.g., but not limited to, liquid, gas,
suspension, aerosols, cell culture medium, and/or biological
fluid). In some embodiments, the fluid can comprise one or more
target species, e.g., but not limited to cells, and/or active
agents described herein. Without wishing to be bound by theory, the
fluid can be liquid (e.g., aqueous or non-aqueous), supercritical
fluid, gases, solutions, and suspensions.
[0303] As used herein, the term "small molecules" refers to natural
or synthetic molecules including, but not limited to, peptides,
peptidomimetics, amino acids, amino acid analogs, polynucleotides,
polynucleotide analogs, aptamers, nucleotides, nucleotide analogs,
organic or inorganic compounds (i.e., including heteroorganic and
organometallic compounds) having a molecular weight less than about
10,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 5,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 1,000
grams per mole, organic or inorganic compounds having a molecular
weight less than about 500 grams per mole, and salts, esters, and
other pharmaceutically acceptable forms of such compounds.
[0304] The term "therapeutic agents" is art-recognized and refers
to any chemical moiety that is a biologically, physiologically, or
pharmacologically active substance that acts locally or
systemically in a subject. Examples of therapeutic agents, also
referred to as "drugs", are described in well-known literature
references such as the Merck Index, the Physicians Desk Reference,
and The Pharmacological Basis of Therapeutics, and they include,
without limitation, medicaments; vitamins; mineral supplements;
substances used for the treatment, prevention, diagnosis, cure or
mitigation of a disease or illness; substances which affect the
structure or function of the body; or pro-drugs, which become
biologically active or more active after they have been placed in a
physiological environment. Various forms of a therapeutic agent may
be used which are capable of being released from a composition into
adjacent tissues or fluids upon administration to a subject.
Examples include steroids and esters of steroids (e.g., estrogen,
progesterone, testosterone, androsterone, cholesterol,
norethindrone, digoxigenin, cholic acid, deoxycholic acid, and
chenodeoxycholic acid), boron-containing compounds (e.g.,
carborane), chemotherapeutic nucleotides, drugs (e.g., antibiotics,
antivirals, antifungals), enediynes (e.g., calicheamicins,
esperamicins, dynemicin, neocarzinostatin chromophore, and
kedarcidin chromophore), heavy metal complexes (e.g., cisplatin),
hormone antagonists (e.g., tamoxifen), non-specific (non-antibody)
proteins (e.g., sugar oligomers), oligonucleotides (e.g., antisense
oligonucleotides that bind to a target nucleic acid sequence (e.g.,
mRNA sequence)), peptides, proteins, antibodies, photodynamic
agents (e.g., rhodamine 123), radionuclides (e.g., I-131, Re-186,
Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-67 and
Cu-64), toxins (e.g., ricin), and transcription-based
pharmaceuticals, anti-inflammatory agents, vaccines, and any
combinations thereof.
[0305] As used herein, the term "molecular toxin" refers to a
compound produced by an organism which causes or initiates the
development of a noxious, poisonous or deleterious effect in a host
presented with the toxin. Such deleterious conditions may include
fever, nausea, diarrhea, weight loss, neurologic disorders, renal
disorders, hemorrhage, and the like. Toxins include, but are not
limited to, bacterial toxins, such as cholera toxin, heat-liable
and heat-stable toxins of E. coli, toxins A and B of Clostridium
difficile, aerolysins, and hemolysins; toxins produced by protozoa,
such as Giardia; toxins produced by fungi. Molecular toxins can
also include exotoxins, i.e., toxins secreted by an organism as an
extracellular product, and enterotoxins, i.e., toxins present in
the gut of an organism.
[0306] As used herein, the term "cells" refers to biological cells
selected from the group consisting of living or dead cells
(prokaryotic and eukaryotic, including mammalian), viruses,
bacteria, fungi, yeast, protozoan, microbes, and parasites. The
biological cells can be a normal cell or a diseased cell. Mammalian
cells include, without limitation; primate, human and a cell from
any animal of interest, including without limitation; mouse,
hamster, rabbit, dog, cat, domestic animals, such as equine,
bovine, murine, ovine, canine, and feline. In some embodiments, the
cells can be derived from a human subject. In other embodiments,
the cells are derived from a domesticated animal, e.g., a dog or a
cat. Exemplary mammalian cells include, but are not limited to,
stem cells, progenitor cells, immune cells, blood cells, and any
combinations thereof. The cells can be derived from a wide variety
of tissue types without limitation such as; hematopoietic, neural,
mesenchymal, cutaneous, mucosal, stromal, muscle, spleen,
reticuloendothelial, epithelial, endothelial, hepatic, kidney,
gastrointestinal, pulmonary, cardiovascular, T-cells, and fetus.
Stem cells, embryonic stem (ES) cells, ES-derived cells and stem
cell progenitors are also included, including without limitation,
hematopoietic, neural, stromal, muscle, cardiovascular, hepatic,
pulmonary, and gastrointestinal stem cells. Yeast cells may also be
used as cells in some embodiments described herein. In some
embodiments, the cells can be ex vivo or cultured cells, e.g. in
vitro. For example, for ex vivo cells, cells can be obtained from a
subject, where the subject is healthy and/or affected with a
disease. While cells can be obtained from a fluid sample, e.g., a
blood sample, cells can also be obtained, as a non-limiting
example, by biopsy or other surgical means know to those skilled in
the art.
[0307] Exemplary fungi and yeast include, but are not limited to,
Cryptococcus neoformans, Candida albicans, Candida tropicalis,
Candida stellatoidea, Candida glabrata, Candida krusei, Candida
parapsilosis, Candida guilliermondii, Candida viswanathii, Candida
lusitaniae, Rhodotorula mucilaginosa, Aspergillus fumigatus,
Aspergillus flavus, Aspergillus clavatus, Cryptococcus neoformans,
Cryptococcus laurentii, Cryptococcus albidus, Cryptococcus gattii,
Histoplasma capsulatum, Pneumocystis jirovecii (or Pneumocystis
carinii), Stachybotrys chartarum, and any combination thereof.
[0308] Exemplary bacteria include, but are not limited to: anthrax,
campylobacter, cholera, diphtheria, enterotoxigenic E. coli,
giardia, gonococcus, Helicobacter pylori, Hemophilus influenza B,
Hemophilus influenza non-typable, meningococcus, pertussis,
pneumococcus, salmonella, shigella, Streptococcus B, group A
Streptococcus, tetanus, Vibrio cholerae, yersinia, Staphylococcus,
Pseudomonas species, Clostridia species, Myocobacterium
tuberculosis, Mycobacterium leprae, Listeria monocytogenes,
Salmonella typhi, Shigella dysenteriae, Yersinia pestis, Brucella
species, Legionella pneumophila, Rickettsiae, Chlamydia,
Clostridium perfringens, Clostridium botulinum, Staphylococcus
aureus, Treponema pallidum, Haemophilus influenzae, Treponema
pallidum, Klebsiella pneumoniae, Pseudomonas aeruginosa,
Cryptosporidium parvum, Streptococcus pneumoniae, Bordetella
pertussis, Neisseria meningitides, and any combination thereof.
[0309] Exemplary parasites include, but are not limited to:
Entamoeba histolytica; Plasmodium species, Leishmania species,
Toxoplasmosis, Helminths, and any combination thereof. Other
examples of parasites can include, but are not limited to,
intestinal worms, rabies, toxoplasma, and any combinations
thereof.
[0310] Exemplary viruses include, but are not limited to, HIV-1,
HIV-2, hepatitis viruses (including hepatitis B and C), Ebola
virus, West Nile virus, and herpes virus such as HSV-2, adenovirus,
dengue serotypes 1 to 4, ebola, enterovirus, herpes simplex virus 1
or 2, influenza, Japanese equine encephalitis, Norwalk, papilloma
virus, parvovirus B19, rubella, rubeola, vaccinia, varicella,
Cytomegalovirus, Epstein-Barr virus, Human herpes virus 6, Human
herpes virus 7, Human herpes virus 8, Variola virus, Vesicular
stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C
virus, Hepatitis D virus, Hepatitis E virus, poliovirus,
Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B,
Measles virus, Polyomavirus, Human Papilomavirus, Respiratory
syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps
virus, Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola
virus, Marburg virus, Lassa fever virus, Eastern Equine
Encephalitis virus, Japanese Encephalitis virus, St. Louis
Encephalitis virus, Murray Valley fever virus, West Nile virus,
Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C,
Sindbis virus, Human T-cell Leukemia virus type-1, Hantavirus,
Rubella virus, Simian Immunodeficiency viruses, and any combination
thereof.
[0311] As used herein, the term "environmental contaminants or
pollutants" refers to microorganisms, molecules, and/or substances
originated from an environmental source, e.g., air, food, and/or
water, that can adversely affect at least one physiological
function of a subject in a temporary or permanent manner, and/or
can be fatal when they are ingested or inhaled by the subject.
Exemplary environmental contaminants or pollutants can include, but
are not limited to microorganisms (e.g., Cryptosporidium, Giardia
lamblia, bacteria, Legionella, Coliforms, viruses, fungi, molds,
spores), bromates, chlorites, haloactic acids, trihalomethanes,
chloramines, chlorine, chlorine dioxide, antimony, arsenic, mercury
(inorganic), nitrates, nitrites, selenium, thallium, Acrylamide,
Alachlor, Atrazine, Benzene, Benzo(a)pyrene (PAHs), Carbofuran,
Carbon, etrachloride, Chlordane, Chlorobenzene, 2,4-D, Dalapon,
1,2-Dibromo-3-chloropropane (DBCP), o-Dichlorobenzene,
p-Dichlorobenzene, 1,2-Dichloroethane, 1,1-Dichloroethylene,
cis-1,2-Dichloroethylene, trans-1,2-Dichloroethylene,
Dichloromethane, 1,2-Dichloropropane, Di(2-ethylhexyl) adipate,
Di(2-ethylhexyl) phthalate, Dinoseb, Dioxin (2,3,7,8-TCDD), Diquat,
Endothall, Endrin, Epichlorohydrin, Ethylbenzene, Ethylene
dibromide, Glyphosate, Heptachlor, Heptachlor epoxide,
Hexachlorobenzene, Hexachlorocyclopentadiene, Lead, Lindane,
Methoxychlor, Oxamyl (Vydate), Polychlorinated, biphenyl s (PCBs),
Pentachlorophenol, Picloram, Simazine, Styrene,
Tetrachloroethylene, Toluene, Toxaphene, 2,4,5-TP (Silvex),
1,2,4-Trichlorobenzene, 1,1,1-Trichloroethane,
1,1,2-Trichloroethane, Trichloroethylene, Vinyl chloride, and
Xylenes, smog, carbon monoxide, sulfur dioxide, and any
combinations thereof.
[0312] By the term "nanomaterials" is generally meant structures
having at least one dimension in nanometer range. Examples of
nanomaterials that can be exposed to cells can include, but are not
limited to, single or multi-walled nanotubes, nanowires, nanodots,
quantum dots, nanorods, nanocrystals, nanotetrapods, nanotripods,
nanobipods, nanoparticles, nanosaws, nanosprings, nanoribbons,
nanocapsules, branched nanomaterials, and any combinations thereof.
The nanomaterials can be made of or comprise, e.g., carbon, metal
or alloy, metal oxide, polymer, or any synthetic materials. In some
embodiments, the nanomaterials can comprise an active agent
described herein, e.g., active agent-loaded nanoparticles.
[0313] As used herein, the term "particulates" refers to fine
particles, powder, flakes, etc., that exist in a relatively small
form. In some embodiments, the particulates are small enough to be
inhaled by a subject. In some embodiments, the particulates can be
produced as a by-product of a chemical reaction, e.g., burning a
fuel. In some embodiments, the particulates can be produced by, for
example, grinding, shredding, fragmenting, pulverizing, atomizing,
or otherwise subdividing a larger form of the material into a
relatively small form. Examples of particulates can include, but
are not limited to, soot, dust, smoke, aerosols, and any
combinations thereof.
[0314] As used herein, the term "aerosols" refers to gaseous
suspensions or solutions of dispersed solid or liquid particles,
e.g., but not limited to, sprays, colloids, mists and respirable
aerosols. The aerosols can be in suspension, solution or dry powder
form. An aerosol can comprise an active agent described herein,
e.g., cells (including, e.g., but not limited to, bacteria and/or
virus), proteins, peptides, antigens, antibodies or portions
thereof, enzymes, nucleic acids, siRNA, shRNA, aptamers, small
molecules, antibiotics, therapeutic agents, molecular toxins,
nanomaterials, particulates, environmental contaminants or
pollutants (e.g., but not limited to, microorganisms,
organic/inorganic contaminants present in food and/or water, and/or
air pollutants). As used herein, the term "respirable aerosol"
refers to an aerosol having a component that can be delivered to
the lungs of a subject, and/or be deposited on the surfaces of a
subject's nasal passages and/or adsorbed onto the tissue of the
nasal passages.
[0315] As used herein, the term "treating" refers to therapeutic
treatment wherein the purpose is to prevent or slow the development
of the disease or disorder. Treatment is generally "effective" if
one or more symptoms or clinical markers are reduced.
Alternatively, a treatment is "effective" if the progression of a
disease is reduced or halted. That is, "treatment" includes not
just the improvement of symptoms or decrease of markers of the
disease, but also a cessation or slowing of progress or worsening
of a symptom that would be expected in absence of treatment.
Beneficial or desired clinical results include, but are not limited
to, alleviation of one or more symptom(s), diminishment of extent
of disease, stabilized (e.g., not worsening) state of disease,
delay or slowing of disease progression, amelioration or palliation
of the disease state, and remission (whether partial or total),
whether detectable or undetectable. "Treatment" can also mean
prolonging survival as compared to expected survival if not
receiving treatment. Those in need of treatment include those
already diagnosed with a disease or disorder, as well as those
likely to develop a disease or disorder.
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specification and examples are expressly incorporated herein by
reference for all purposes. These publications are provided solely
for their disclosure prior to the filing date of the present
application. Nothing in this regard should be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention or for any other reason.
All statements as to the date or representation as to the contents
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applicants and does not constitute any admission as to the
correctness of the dates or contents of these documents.
* * * * *
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