U.S. patent application number 16/358160 was filed with the patent office on 2019-12-12 for microgels and microtissues for use in tissue engineering.
The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Sangeeta N. BHATIA, Cheri Y. LI.
Application Number | 20190376024 16/358160 |
Document ID | / |
Family ID | 46210418 |
Filed Date | 2019-12-12 |
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United States Patent
Application |
20190376024 |
Kind Code |
A1 |
BHATIA; Sangeeta N. ; et
al. |
December 12, 2019 |
MICROGELS AND MICROTISSUES FOR USE IN TISSUE ENGINEERING
Abstract
The present invention features microgels and microtissues for
use in tissue engineering. Featured is a microencapsulation device
for making microgels and/or microtissues via an emulsion
technology. Also featured are methods of making higher ordered
structures that mimic in vivo tissue structures. Methods of us are
also featured.
Inventors: |
BHATIA; Sangeeta N.;
(Lexington, MA) ; LI; Cheri Y.; (Cambridge,
MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
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Family ID: |
46210418 |
Appl. No.: |
16/358160 |
Filed: |
March 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14116901 |
Apr 14, 2014 |
10260039 |
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PCT/US2012/037656 |
May 11, 2012 |
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16358160 |
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61484987 |
May 11, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/02 20130101; C12N
2531/00 20130101; C12N 2533/30 20130101; C12N 5/0671 20130101; C12N
5/0062 20130101; C12N 2513/00 20130101; C12M 23/16 20130101; C12N
5/0012 20130101 |
International
Class: |
C12N 5/00 20060101
C12N005/00; C12N 5/071 20060101 C12N005/071; C12Q 1/02 20060101
C12Q001/02 |
Goverment Interests
STATEMENT OF FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
nos. ROI-DK56966 6914791 and ROI-EB008396 6920502, awarded by the
National Institutes of Health (NIDDK). The government has certain
rights in the invention.
Claims
1. A population of microtissues produced by a method comprising
injecting into a channel of a microfluidic device a first input
stream comprising a solution of cells and a second input stream
comprising a polymerizable hydrogel solution to form a combined
aqueous stream; emulsifying the combined aqueous stream with an
emulsion stream in a first region of the channel of the
microfluidic device, to produce droplets comprising the cells in
the polymerizable hydrogel, mixing said droplets to disperse the
cells in the polymerizable hydrogel in a second region of the
channel comprising a serpentine section in the microfluidic device;
polymerizing the droplets comprising the cells dispersed in the
polymerizable hydrogel to form the microtissues; and collecting the
microtissues from an outlet of the device, such that the population
of microtissues is made.
2. The population of claim 1, wherein the hydrogel material is
agarose, fibrin, or polyethylene hydrogel.
3. A population of microtissues produced by a method comprising
injecting into a channel of a microfluidic device a solution
comprising pre-stabilized, micropatterned cell clusters and a
polymerizable hydrogel solution, wherein the cell clusters comprise
parenchymal cells and supporting nonparenchymal cells; emulsifying
the solution of cell clusters and polymerizable hydrogel with an
emulsion stream in a channel of the microfluidic device, to produce
droplets comprising the cells in the polymerizable hydrogel,
polymerizing the droplets comprising the cells dispersed in the
polymerizable hydrogel to form the microtissues, wherein
polymerizing occurs during transport of the droplets, wherein
transport occurs continuously; and collecting the microtissues from
an outlet of the device, such that the population of microtis sues
is made.
4. The population of claim 3, wherein the cell clusters comprise
primary hepatocytes and stromal cells, for example,
fibroblasts.
5. The population of claim 3, wherein the cell clusters comprise
hepatocytes selected from the group consisting of
progenitor-derived hepatocytes, ES-derived hepatocytes, and induced
pluripotent stem cell-derived (iPS-derived) hepatocytes, and
stromal cells, for example, fibroblasts.
6. The population of claim 3, wherein the cell clusters comprise
cancer cells and stromal cells, for example, fibroblasts.
7. The population of claim 1, wherein the prepolymerized hydrogel
is a photopolymerizable hydrogel, for example, polyethylene glycol
(PEG) hydrogel.
8. The population of claim 1, wherein the hydrogel is
functionalized with one or more affinity biomolecules facilitating
higher ordered assembly of the said microtissues.
9. The population of claim 8, wherein the biomolecule is
streptavidin, or a cell adhesive peptide, for example, the
biomolecule is RGDS peptide.
10. The population of claim 3, wherein the droplets or microtissues
are about 50 to about 250 .mu.M in diameter.
11. The population of claim 1, wherein the droplets of microtissues
are about 20 to about 150 .mu.M in diameter.
12. The population of claim 1, wherein the droplets comprise about
1 to about 50 cells.
13. The population of claim 1, wherein each microtissue comprises
about 2 to about 20 cells or wherein each microtissue comprises
about 5 to about 10 cells.
14. A method of making a construct, comprising assembling all or a
portion of the population of microtissues of claim 1 into a higher
ordered structure.
15. The method of claim 14, wherein the microtissues comprise an
encoding biomolecule or affinity ligand and a substrate is
patterned with a templating biomolecule or ligand, such that when
the substrate is contacted with the population of microtissues, the
microtissues are assembled in the pattern of the substrate.
16. The method of claim 15, wherein the encoding and templating
biomolecules are complementary DNAs or complementary affinity
ligands.
17. The method of claim 14, wherein the microtissues are assembled
by physical means.
18. A construct produced by the method of claim 15.
19. An assay system featuring the construct of claim 18.
20. The assay system of claim 19, which is a metabolic assay system
or a toxicology assay system.
21. The assay system of claim 20, which is a screening assay system
for anti-tumorogenic compounds.
22. A microencapsulation device comprising one or more injection
ports in contact with a microchannel comprised therein, said
injection ports for introducing one or more aqueous solutions, a
droplet generating nozzle into which said solutions flow, an
emulsion stream in contact with said droplet generating nozzle, a
mixer section for dispersing components of droplets, a polymerizing
section, and an outlet.
23. A microencapsulation device as depicted in FIG. 2.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/116,901, filed Apr. 14, 2014, now pending,
which application is a 35 U.S.C. .sctn. 371 National Stage Entry of
PCT Application No. PCT/US2012/037656, filed on May 11, 2012, which
claims the benefit of the priority date of U.S. Provisional
Application No. 61/484,987, which was filed on May 11, 2011. The
entire content of each of the above-referenced patent applications
is hereby incorporated by reference in its entireties.
REFERENCE TO SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format via EFS-Web and
is hereby incorporated by reference in its entirety. Said ASCII
copy, created Feb. 27, 2019, is named
"MITY-012USDV_Sequence-Listing.txt" and is 2146 Kilobytes in
size.
SUMMARY OF THE INVENTION
[0004] The present invention features microgels and microtissues
for use in tissue engineering. In particular the present invention
features methods for fabricating polymerized hydrogels on a
microscale. Microgels and/or microtissues of the present invention
are capable of being assembled into larger ordered constructs which
resemble or mimic in vivo tissue architecture. The microgels and/or
microtissues of the invention are made using devices engineered to
produce microscale droplets comprising living cells and/or other
biologically relevant compounds wherein the droplets comprise, for
example, a photopolymerizable hydrogel or other suitable scaffold
component. During assembly, the droplets are made, mixed, and
subsequently polymerized to form the microgels or microtis sues of
the invention. In preferred embodiments, the hydrogel components
(or other scaffold components) are chemically modified or
chemically modifiable in order to provide cell supportive or other
assembly promoting properties. Microgels and/or microtissues of the
present invention are particularly suitable for use in generating
higher ordered structures. For example, the microgels and/or
microtissues can be assembled on surfaces or substrates e.g.,
slides, tissue culture substrates, and the like, or can be
assembled into larger three-dimensional structures for use in a
variety of tissue engineering applications.
[0005] Described herein is an approach for the patterning and
assembly of engineered tissues from microtissues, small units
(<1 mm) of cell-laden hydrogels, programmed by the hybridization
of single-stranded DNA oligonucleotides. Also described herein are
approaches for the patterning and assembly of engineered tissues
from microtis sues in combination with microgels, e.g., microgels
comprising biologically relevant compounds. Further described
herein are alternative means for assembling the microtissues and/or
microgels of the invention in order to create engineered tissues.
This platform will have general utility for constructing in vitro
disease models and commercial applications in biomedical tissue
manufacture.
BRIEF DESCRIPTION OF TH DRAWINGS
[0006] FIG. 1. Schematic of microtissue encapsulation,
functionalization, and DNA-templated self assembly. Cells are
injected with a photopolymerizable hydrogel prepolymer into a
high-throughput microfluidic encapsulation device. Droplets of the
cell-prepolymer mixture are exposed to UV on-chip to form
streptavidin-containing microtissues which are then coated with
5'-biotin terminated oligonucleotides. Encoded microtissues
containing different cell types are seeded on a DNA microarray
template which directs the binding of microtis sues to specific
spots on the templating surface, attaining sequential DNA-templated
patterning of cell-laden microtis sues.
[0007] FIGS. 2A-2F. Microencapsulation device. (FIG. 2A) Overview
of device showing two aqueous input streams (red, blue) dispersed
by shear flow from an oil stream into droplets that mix (purple)
and travel down the UV-exposure channel. (FIG. 2B) Prepolymer
(2.times. concentrated) and a cell suspension meet and flow into a
60 .mu.m droplet generating nozzle. Vertical columns on either side
of the channel provide visual references (50-100 .mu.m below,
100-150 .mu.m above) for real-time adjustment of droplet size.
(FIG. 2C) Droplets pass through a bumpy serpentine mixer section to
thoroughly disperse cells in prepolymer and are then polymerized by
UV irradiation from a curing lamp. (FIG. 2D) Microtissues collected
from the device (6000/min) are spherical and monodisperse. (FIG.
2E) Microtissue size is controlled by the relative flow rates of
the combined aqueous phase (Q.sub.P) and the continuous oil phase
(Q.sub.O), and increases with prepolymer: oil flow ratio. (FIG. 2F)
Adding small amounts of Krytox 157 FSH fluorosurfactant into the
oil decreased droplet diameter at all flow ratios, allowing higher
prepolymer flow rates for a given microtissue size.
[0008] FIGS. 3A-3D. Microtissue functionalization. (FIG. 3A) The
primary hydrogel component, acrylate-PEG20k-acrylate macromonomer,
was mixed with conjugated acrylate-PEG-streptavidin (0-2 mg/ml)
before photo-initiated free radical polymerization, forming a
hydrogel network that is decorated with pendant streptavidin
proteins. (FIG. 3B) PEG-streptavidin microtissues stained with
biotin-4-fluorescein, which can freely diffuse through the hydrogel
network, and anti-streptavidin IgG which is restricted to the
surface of the microtissues. The intensity of biotin-4-fluorescein
staining increased linearly with the bulk concentration of
covalently-bound streptavidin, while antibody stains for surface
concentration increased only as a power of bulk concentration.
(FIG. 3C) PEG-SA microtis sues are further functionalized with
biotin-ssDNA. The availability of this ssDNA to hybridize with a
templating surface was tested using 1 .mu.m fluorescent beads
coated with DNA. (FIG. 3D) Microtissues with the appropriate
complementary sequence were coated with hybridized beads. No beads
hybridized to control-sequence microtissues, which remained dark in
the green channel and showed only encapsulated marker beads in the
phase image.
[0009] FIGS. 4A-4H. Capture efficiency and specificity of
DNA-directed microtissue assembly. (FIG. 4A) Number of
DNA-functionalized microtissues containing fluorescent beads as
markers captured on microarray spots with increasing spotting
concentration of complementary oligonucleotide. (FIG. 4B)
Quantified assembly results from microtissues seeded over an array
of complementary spots at both high (shown on the left) and low %
surface coverage. Control arrays of noncomplementary spots remained
blank. Capture efficiency is calculated as the ratio of capture
density to seeding density. (FIG. 4C) Three-color (RGB) microtissue
assembly using a set of orthogonal oligonucleotide sequences: B
(red), C (green), and D (blue). Microtissues contain encapsulated
marker beads. (FIG. 4D) Quantified percentages of microtissues on
target spots (1 column) vs. off-target spots (2 columns). (FIG. 4E)
MIT logo assembled in microtissues of C (green) and D (blue), and
(FIG. 4F) photograph of templating slide illustrating scale of
assembled microtissue patterns.
[0010] FIGS. 5A-5F. Cell encapsulation and microtissue culture.
(FIG. 5A) Rat fibroblast (J2-3T3) and human lymphoblast (TK6) cell
lines uniformly encapsulated within microtissues and stained for
viability. (FIG. 5B) Histogram of J2-3T3 5 distribution within
microtissues and comparison to optimal Poisson statistics. (FIG.
5C) Viability of J2-3T3 and TK6 cells three hours
post-encapsulation at increasing % UV overexposure past the minimum
intensity required to fully polymerize microtissues. (FIG. 5D)
J2-3T3 cells attached and spread within microtissues decorated with
RGDS (SEQ ID NO: 1) peptides. (FIG. 5E) Human lung adenocarcinoma
(A549) cells aggregated to form multicellular tumor spheroids
within microtissues. (FIG. 5F) Microtissues encapsulating either
J2-3T3 (CellTracker Green) or A549 cells (CellTracker Blue) were
self-assembled into composite hexagonal clusters.
[0011] FIG. 6. Optimization of acrylate-PEG-streptavidin
conjugation. Non-denaturing PAGE gel (top) of purified products
from varying molar ratios of reactants. At low ratios, discrete
bands of protein with 1-5 modified amines are visible. At higher
ratios, streptavidin is over-modified and biotin-binding capacity
is significantly reduced. Reaction conditions of interest were
further tested by incorporating products into microtissues, binding
biotin-DNA, and staining by hybridization with DNA-coated beads
(bottom).
[0012] FIGS. 7A-7B. Distribution of cell encapsulation numbers
within microtissues. (FIG. 7A) Prior to process modifications,
cells that were suspended in prepolymer settled within tubing
between the syringe and the device, resulting in oscillating cell
density reaching the nozzle and an uneven number of cells per
microtissue. (FIG. 7B) When cells are injected in an isopycnic
medium, and as a separate stream from concentrated prepolymer, the
distribution narrowed to the Poisson limit.
[0013] FIGS. 8A-8C. Multi-photon images of fibroblast spreading
within RGDS microtissues. (FIG. 8A) Maximum intensity projection
and (FIG. 8B-8C) slice images of J2-3T3 fibroblasts spreading on
Day 4 post-encapsulation. Red: actin (phalloidin), green: hydrogel
(biotin-4-fluorescein), bright-green: nuclei (Hoecht).
[0014] FIG. 9. Fibroblast-laden, RGD-decorated microtissues
cultured in close contact and in the presence of non-encapsulated
fibroblasts. Contiguous microtissue-assembled structures linked by
adherent cells formed by D1 post-encapsulation.
[0015] FIG. 10A) Albumin secretion as a measure of hepatic
phenotype, elevated in the presence of fibroblasts and RGDS
peptide. FIG. 10B) Luminex bead measurements of human drug
metabolism enzyme genes, showing upregulation of the genes in 3D as
opposed to 2D, in various phases of drug metabolism. FIG. 10C)
Demonstrated CYP450 activity in 3D engineered hepatic tissues,
including drug-drug interactions.
[0016] FIG. 11. Extended albumin production in the presence of J2's
by pre-patterning primary hepatocytes into clusters with cell-cell
contacts prior to encapsulation.
[0017] FIG. 12A. Hepatic microtissues (.about.150 um diameter)
generated by droplet-based encapsulation device, living hepatocytes
stained in green. FIG. 12B) Hepatic microtissues respond to
hepatotoxic compounds such as acetaminophen (increased red-dead
signal).
[0018] FIG. 13. Membrane perfusion format.
[0019] FIG. 14. Depicts a large-particle flow sort and analyzer.
Depicted is a split-injection encapsulation chip with a
large-particle flow analyzer and sorter (COPAS Biosort by Union
Biometrica). Cells can be encapsulated within microtissues that can
contain tumor cells, stromal cells, and various entrapped ECM.
These microtissues can then be further enriched using the flow
sorter that measures the fluorescence intensity each
microtissue
[0020] FIG. 15A) Homotypic control: microtissue populations sorted
into three bins (low-green, medium-pink, and high-blue) by
flow-analyzer fluorescence correlate closely with average number of
encapsulated cells per microtissue. FIG. 15B) Using fluorescence as
a measure of cell density, the histogram of each microtis sue
population distinctly shifts right during culture as cells
proliferate. (FIGS. 15C-15D) Heterotypic control: microtissues
containing two cell types labeled green or far-red can be sorted
into user-defined bins such as low stromal/tumor cell ratio (FIG.
15C) or high stromal/tumor ratio (FIG. 15D).
[0021] FIGS. 16A-16B. Microtissue proliferation modiulated by (FIG.
16A) incorporated extracellular matrix molecules, or (FIG. 16B)
culture in soluble growth factors.
[0022] FIGS. 17A-17C. Differences in tumor cell responses to small
molecules in 2D vs. 3D culture. (FIG. 17A) Comparison of
proliferation rates on 2D-microplate and 3D-microtis sue formats
relative to untreated controls. (FIG. 17B) Growth curve over time
of tumor cells in 2D measured by microplate fluorescence, and (FIG.
17C) Growth curve over time of tumor cells in 3D measured by gel
dissolution and DNA quantitation.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention provides methods of encapsulating cells or
other biologically relevant molecules within microtissues or
mocrogels on a microfluidic device, functionalizing the
microtissues or microgels with an affinity ligand, e.g. with
single-stranded DNA, and selectively binding microtissues (e.g.,
affinity ligand encoded, e.g., DNA encoded) to a variety of
surfaces functionalized with the a complementary ligand (e.g., a
templating lignad, e.g., complementary single-stranded DNA),
including patterns on a templating slide or other
hydrogels/microtissues. In exemplary aspects of the invention,
cells are injected as a suspension in isopycnic medium into a cell
encapsulation device. A photopolymerizable hydrogel, containing
biotin-binding groups, is injected into the device simultaneously,
and the two streams mix on-chip. The combined aqueous stream is
then sheared by intersecting oil streams into uniform droplets,
which are gelled through UV irradiation. Resulting microtissues are
incubated with biotin-terminated single-stranded DNA to provide
microtissue surfaces with specific recognition code. This code
directs microtissue binding and assembly based on base-pair binding
with complementary sequences.
[0024] In exemplary methods, encoded microtis sues are assembled
onto DNA-patterned templates, composed of microarrayed DNA spots on
a glass slide. Encoded microtissues are seeded onto microarray
slides, either in a concentrated slurry or by settling from a
dilute suspension, and quickly bind (<10 minutes) to only target
spots. Microtissues not bound to target spots are washed off, and
the process is repeated with microtissues coated with other
sequences (potentially containing different cell types,
biomolecules, etc.) to sequentially build patterns of microtis sue
types. We further envision that DNA-directed assembly can be used
to bind additional layers of microtis sues upon this initial
surface-templated layer.
[0025] Fundamentally, living tissues are composed of multiple cell
types organized with microscale architectures. Current tools to
specify heterostructures of cells include dielectrophoresis,
molding (e.g. aggregates, cords), ECM-patterning, and
DNA-modification of cells to direct assembly (Hsiao, S. C.; Shum,
B. J.; Onoe, H.; Douglas, E. S.; Gartner, Z. J.; Mathies, R. A.;
Bertozzi, C. R.; Francis, M. B. "Direct cell surface modification
with DNA for the capture of primary cells and the investigation of
myotube formation on defined patterns" Langmuir 2009, 25,
6985-6991. Gartner, Z. J.; Bertozzi, C. "Programmed assembly of
three-dimensional microtissues with defined cellular connectivity"
Proc. Natl. Acad. Sci. 2009, 106, 4606-4610). DNA-modified cells
can be effectively assembled using orthogonal pairs of
DNA-sequences, but the method has been demonstrated only for
limited cell types. Furthermore, encoding DNA is bound directly
onto cell membranes where covalently bound ligands may be
susceptible to recycling and may potentially modify cell function.
In general, current approaches focus on developing novel techniques
to position individual cells, which can be then immobilized within
a scaffold. This invention calls for a novel, inverse strategy of
pre-encapsulating cells within a scaffold, and the subsequent
positioning of these microtis sues by DNA-hybridization.
[0026] Several non-obvious aspects of this invention address unique
challenges in extending DNA-directed assembly from cells to
microtissues. Until now, DNA-templated assembly has not been
applied to larger units such as microtissues (100 um), which
present inherent difficulties in mass transport (gravity becomes a
dominant factor in the ability of surfaces to sufficiently
interact) and binding (stronger washing forces on large objects
require a larger number of DNA-hybridization bonds). To compensate
the problem of increasing microtis sue size, our method strictly
controls the size of microtissues fabricated, and optimizes
microtis sue DNA-functionalization (using streptavidin-biotin
binding) and template spotting to achieve high surface densities.
Streptavidin-biotin based DNA-functionalization of microtissues is
simple, modular, and cytocompatible.
[0027] Post-encoding of microtissues with biotin-DNA avoids UV
damage that would occur by premixing DNA into the polymer, and
allows the same batch of microtissues to be labeled after culture
in various conditions. Other bioconjugation methods exist to modify
hydrogel networks post-encapsulation (e.g. maleimide or NHS
chemistries) but often require reaction conditions that are
incompatible with maintaining the viability of encapsulated cells.
Finally, the controlled spherical shape of droplet-derived microtis
sues minimizes contact area with templating surfaces, drastically
reducing non-specific adhesion, and allowing templates to be made
with a range of materials/chemistries (e.g. hydrophilic-glass vs.
hydrophobic-PDMS). Spherical microtissues provide maximum surface
area to volume for faster diffusion of nutrients to encapsulated
cells, and can be assembled into isotropic structures (e.g. perfect
close packing).
[0028] There are many advantages associated with patterning
cellular microtis sues rather than individual cells. First, cells
are pre-encapsulated in a modular synthetic scaffold that can be
easily customized with degradable linkages, adhesive ligands, and
other biologically or chemically active factors. For sensitive
cells such as primary hepatocytes, stabilization within a tailored
3D environment is necessary to promote their growth and function.
Second, microtissues containing one cell type can be first cultured
separately to stabilize homotypic interactions before they are
assembled with other microtissues to activate heterotypic
interactions. Third, because DNA is bound to the hydrogel scaffold
rather than cell membranes, encoded microtissues can remain in
assembled patterns for an extended period of time without
additional measures for immobilization (e.g. embedding in agarose),
and then removed for further culture, isolation, and biochemical
analysis. These two advantages provide an additional layer of
flexibility in studying the temporal aspects of intercellular
signaling processes.
[0029] So that the invention may be more readily understood,
certain terms are first defined.
[0030] As used herein, the term "microtissue" refers to a
microscale polymerized hydrogel or other suitable scaffold which
comprises living cells. As used herein, the term "microgel" refers
to a microscale polymerized hydrogel or other suitable scaffold
which comprises one or more biologically relevant compounds or
agents. As used herein, the term microscale refers to objects
having a size, e.g., a two-dimensional or three-dimensional feature
size less than 1 mm. For example microscale objects can have a
length and/or width and/or height and/or diameter or other feature
size of greater than 1 .mu.m and less than 1 mm. In preferred
embodiments, microscale objects, e.g., microtissues and/or
microgels of the invention have a size e.g., a diameter of 10 to
500 .mu.m, 20 to 400 .mu.m, 25 to 300 .mu.m, or 50 to 250
.mu.m.
[0031] As used herein the term "construct" refers to a higher
ordered assembly of the microgels and/or microtis sues of the
invention. A construct can be assembled, for example, using the DNA
directed assembly methods of the invention, using physical packing,
or using other suitable chemical means of physically assembling the
microgels and/or microtis sues of the invention.
[0032] As used herein the term "on-chip emulsion" or "on-chip
emulsification" refers to the generation of microtissues and/or
micro gels of the invention using the physical devices, i.e.,
chips, defined herein.
[0033] As used herein, the term "co-culture" refers to a collection
of cells cultured in a manner such that more than one population of
cells are in association with each other. Co-cultures can made such
that cells exhibit heterotypic interactions (i.e., interaction
between cells of populations of different cell types), homotypic
interactions (i.e., interaction between cells of the same cell
types) or co-cultured to exhibit a specific and/or controlled
combination of heterotypic and homotypic interactions between
cells.
[0034] As used herein, the term "pre-mixing" refers to a mixing of
cells and/or other biologically relevant components in a
prepolymerized hydrogel or scaffold material in a manner such that
components within the population are distributed, e.g., evenly
distributed throughout the prepolymerized hydrogel or scaffold
material.
[0035] As used herein, the term "encapsulation" refers to the
confinement of a cell or population of cells within a material, in
particular, within a biocompatible polymeric scaffold or hydrogel.
The term "co-encapsulation" refers to encapsulation of more than
one cell or cell type or population or populations of cells within
the material, e.g., the polymeric scaffold or hydrogel.
[0036] As used herein, the term "biochemical factor" or
"biochemical cue" refers to an agent of a chemical nature having a
biological activity, for example, on a cell or in a tissue.
Exemplary biochemical factors or cues include, but are not limited
to growth factors, cytokines, nutrients, oxygen, proteins,
polypeptides and peptides, for example, adhesion-promoting
proteins, polypeptides and peptides, and the like. Exemplary
adhesion-promoting peptides include those derived from the
extracellular matrix (ECM) of a cell or tissue, including, but not
limited to collagen-derived peptides, laminin-derived peptides,
fibronectin-derived peptides (e.g., the RGD-peptides), and the
like.
[0037] As used here in, the term "affinity biomolecules" refers to
a biomolecule e.g., a protein, peptide, nucleic acid molecule, or
the like, suitable for affinity binding to other compatible
biomolecules. In exemplary embodiments of the invention, the
affinity biomolecule is streptavidin in which is suitable for
affinity binding to biotin. In other exemplary embodiments of the
invention, the affinity biomolecule is a peptide, e.g., a cell
adhesive peptide, suitable for affinity binding to compatible cell
surface biomolecules.
[0038] Co-cultures can be maintained in vitro or can be included in
engineered tissue constructs of the invention, maintained in vitro
and/or implanted in vivo.
[0039] As used herein, the term "hydrogel" refers to a network of
polymer chains that are hydrophilic in nature, such that the
material absorbs a high volume of water or other aqueous solution.
Hydrogels can include, for example, at least 70% v/v water, at
least 80% v/v water, at least 90% v/v water, at least 95%, 96%,
97%, 98% and even 99% or greater v/v water (or other aqueous
solution). Hydrogels can comprise natural or synthetic polymers,
the polymeric network often featuring a high degree of
crosslinking. Hydrogels also possess a degree of flexibility very
similar to natural tissue, due to their significant water content.
Hydrogel are particularly useful in tissue engineering applications
of the invention as scaffolds for culturing cells. In preferred
embodiments of the invention, the hydrogels are made of
biocompatible polymers. Hydrogels of the invention can be
biodegradable or non-biodegradable.
[0040] As used here, the term "parenchymal cells" refers to cells
of, or derived from, the parenchyma of an organ or gland, e.g., a
mammalian organ or gland. The parenchyma of an organ or gland is
the functional tissue of the organ or gland, as distinguished from
surrounding or supporting or connective tissue. As such,
parenchymal cells are attributed with carrying out the particular
function, or functions, of the organ or gland, often referred to in
the art as "tissue-specific" function. Parenchymal cells include,
but are not limited to, hepatocytes, pancreatic cells (alpha, beta,
gamma, delta), myocytes, e.g., smooth muscle cells, cardiac
myocytes, and the like, enterocytes, renal epithelial cells and
other kidney cells, brain cell (neurons, astrocytes, glia cells),
respiratory epithelial cells, stem cells, and blood cells (e.g.,
erythrocytes and lymphocytes), adult and embryonic stem cells,
blood-brain barrier cells, adipocytes, splenocytes, osteoblasts,
osteoclasts, and other parenchymal cell types known in the art.
Because parenchymal cells are responsible for tissue-specific
function, parenchymal cells express or secrete certain tissue
specific markers.
[0041] Certain precursor cells can also be included as "parenchymal
cells", in particular, if they are committed to becoming the more
differentiated cells described above, for example, liver progenitor
cells, oval cells, adipocytes, osteoblasts, osteoclasts, myoblasts,
stem cells (e.g., embryonic stem cells, hematopoietic stem cells,
mesenchymal stem cells, endothelial stem cells, and the like. In
some embodiments stem cells can be encapsulated and/or implanted
under specified conditions such that they are induced to
differentiate into a desired parenchymal cell type, for example, in
the construct and/or in vivo. It is also contemplated that
parenchymal cells derived from cell lines can be used in the
methodologies of the invention.
[0042] The term "non-parenchymal cells" as used herein, refers to
the cells of or derived from the tissue surrounding or supporting
parenchymal tissue in an organ or gland, for example, in a
mammalian (e.g., human) organ or gland, or the connective tissue of
such an organ or gland. Exemplary non-parenchymal cells include,
but are not limited to, stromal cells (e.g., fibroblasts),
endothelial cells, stellate cells, cholangiocytes (bile duct
cells), Kupffer cells, pit cells, and the like. The choice of
non-parenchymal cells used in the constructs of the invention will
depend upon the parenchymal cell types used.
[0043] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a cellular island" includes a plurality of such cellular islands
and reference to "the cell" includes reference to one or more cells
known to those skilled in the art, and so forth.
[0044] As used herein the term "encoding biomolecule" of "encoding
affinity ligand" refers to an affinity biomolecules Incorporated
within. or on the surface of, a microgel or microtissue of the
invention. The term "templating biomolecule" or "templating
affinity ligand" refers to affinity biomolecules, e.g.
complementary affinity biomolecules, incorporated on the surface of
a substrate, e.g. a cell culture or tissue culture substrate. As
used herein the term "encoding DNA" refers to DNA incorporated
within or on the surface of microgel or microtissue of the
invention. The term "templating DNA" refers to DNA e.g.,
complimentary DNA Incorporated on the surface of a substrate e.g. a
cell culture or tissue culture substrate.
[0045] Various aspects of the invention are described in further
detail in the following subsections.
I. Cell Sources
[0046] The technology of the instant invention is readily amenable
to use with a variety of cell types including primary cells, cell
lines, transformed cells, precursor and/or stem cells, and the
like. Exemplary embodiments feature use of parenchymal cells,
optionally in combination with non-parenchymal cells, to produce
engineered tissue constructs having differentiated function, e.g.,
for the modeling of primary tissues.
[0047] Parenchymal cells can be obtained from a variety of sources
including, but not limited to, liver, skin, pancreas, neuronal
tissue, muscle, and the like. Parenchymal cells can be obtained
from parenchymal tissue using any one of a host of art-described
methods for isolating cells from a biological sample, e.g., a human
biological sample. Parenchymal cells. e.g., human parenchymal
cells, can be obtained by biopsy or from cadaver tissue. In certain
embodiments, parenchymal cells are derived from lung, kidney,
nerve, heart, fat, bone, muscle, thymus, salivary gland, pancreas,
adrenal, spleen, gall bladder, liver, thyroid, paraythyroid, small
intestine, uterus, ovary, bladder, skin, testes, prostate, or
mammary gland.
[0048] In exemplary aspects, the invention employs constructs
containing human parenchymal cells optimized to maintain the
appropriate morphology, phenotype and cellular function conducive
to use in the methods of the invention. Primary human parenchymal
cells can be isolated and/or pre-cultured under conditions
optimized to ensure that the parenchymal cells of choice initially
have the desired morphology, phenotype and cellular function and,
thus, are poised to maintain said morphology, phenotype and/or
function in the constructs, and in vivo upon implantation to create
the humanized animals of the invention
[0049] Cells useful in the methods of the disclosure are available
from a number of sources including commercial sources. For example,
hepatocytes may be isolated by conventional methods (Berry and
Friend, 1969, J. Cell Biol. 43:506-520) which can be adapted for
human liver biopsy or autopsy material. In general, cells may be
obtained by perfusion methods or other methods known in the art,
such as those described in U.S. Pat. Pub. No. 20060270032.
[0050] Parenchymal and non-parenchymal cell types that can be used
in the above-described constructs include, but are not limited to,
hepatocytes, pancreatic cells (alpha, beta, gamma, delta),
myocytes, enterocytes, renal epithelial cells and other kidney
cells, brain cell (neurons, astrocytes, glia), respiratory
epithelium, stem cells, and blood cells (e.g., erythrocytes and
lymphocytes), adult and embryonic stem cells, blood-brain barrier
cells, and other parenchymal cell types known in the art,
fibroblasts, endothelial cells, and other non-parenchymal cell
types known in the art.
[0051] Typically, in practicing the methods of the disclosure, the
cells are mammalian cells, although the cells may be from two
different species (e.g., humans, mice, rats, primates, pigs, and
the like). The cells can be primary cells, or they may be derived
from an established cell-line. Cells can be from multiple donor
types, can be progenitor cells, tumor cells, and the like. In
preferred embodiments, the cells are freshly isolated cells (for
example, encapsulated within 24 hours of isolation), e.g., freshly
isolated cells from donor organs. In certain embodiments, the cells
are isolated from individual donor organs and an assembled tissue
construct is specific for that donor. Any combination of cell types
that promotes maintenance of differentiated function of the
parenchymal cells can be used in the methods and constructs of the
invention (e.g., parenchymal and one or more populations of
non-parenchymal cells, e.g., stromal cells). Parenchymal cells
which may be cultured in the constructs as described herein may be
from any source known in the art, e.g., primary hepatocytes,
progenitor-derived, ES-derived, induced pluripotent stem cells
(iPS-derived), etc.
[0052] Further cell types which may be cultured in the constructs
of the invention include pancreatic cells (alpha, beta, gamma,
delta), enterocytes, renal epithelial cells, astrocytes, muscle
cells, brain cells, neurons, glia cells, respiratory epithelial
cells, lymphocytes, erythrocytes, blood-brain barrier cells, kidney
cells, cancer cells, normal or transformed fibroblasts, liver
progenitor cells, oval cells, adipocytes, osteoblasts, osteoclasts,
myoblasts, beta-pancreatic islets cells, stem cells (e.g.,
embryonic stem cells, hematopoietic stem cells, mesenchymal stem
cells, endothelial stem cells, etc.), cells described in U.S. pat.
app. Ser. No. 10/547,057 paragraphs 0066-0075 which is incorporated
herein by reference, myocytes, keratinocytes, and indeed any cell
type that adheres to a substrate.
[0053] It is understood that constructs of the invention may
contain parenchymal cells with one, or two or more types of
non-parenchymal cells such as, for example, stromal cells,
endothelial cells, etc. One of skill in the art will appreciate
that particular patterns of non-parenchymal cells and/or
parenchymal cells may be desired in some cases, e.g., when it is
desired to mimic certain in vivo environments. It is understood
that any support or accessory cells may be included in the
constructs of the invention.
[0054] In other exemplary embodiments, the microtissues of the
invention feature immortalized, transformed tumorogenic cells.
Cells can be either primary cells, for example, from a tumor
biopsy, or cell lines. In one embodiment, tumor cells are
encapsulated within a first population of microtissues and stromal
cells are encapsulated in a second population of microtis sues. One
or more populations of mictotis sues can further include, for
example, ECM proteins entrapped therein. Alternatively, ECM
components can be entrapped within separate populations or within a
separate population of microgels. Higher ordered structures can be
created using any combination or these microtissues or
microgels.
II Device
[0055] Microencapsulation devices of the invention are described in
detail in the appended Examples and Figures. In a preferred
embodiment, a microencapsulation device on the invention comprises
one or more injection ports in contact with a microchannel
comprised therein, said injection ports for introducing one or more
aqueous solutions, a droplet generating nozzle into which said
solutions flow, an emulsion stream in contact with said droplet
generating nozzle, a mixer section for dispersing components of
droplets, a polymerizing section, and an outlet.
[0056] A preferred microencapsulation device is depicted in FIG. 2.
The microencapsulation device includes two aqueous input ports into
which aqueous solutions enter and streams are dispersed by shear
flow from an oil stream into droplets that mix and travel down the
UV-exposure channel, comprised in the polymerization section. In an
exemplary embodiment, prepolymer (2.times. concentrated) and a cell
suspension meet and flow into a 60 .mu.m droplet generating nozzle.
Vertical columns on either side of the channel provide visual
references (50-100 .mu.m below, 100-150 .mu.m above) for real-time
adjustment of droplet size. Droplets pass through a mixer section,
e.g., a bumpy serpentine mixer section, to thoroughly disperse
cells in prepolymer which are then polymerized by UV irradiation
from a curing lamp, in the polymerization section. Microtissues are
collected from the device (6000/min) via an outlet and are
spherical and monodisperse. Microtissue size is controlled by the
relative flow rates of the combined aqueous phase (Q.sub.P) and the
continuous oil phase (Q.sub.O), and increases with prepolymer: oil
flow ratio. Adding small amounts of a surfactant, e.g., Krytox 157
FSH fluorosurfactant into the oil decreased droplet diameter at all
flow ratios, allows for higher prepolymer flow rates for a given
microtissue size.
III Methods of Making Microtissues of Microgels
[0057] The present invention features processes by which to
encapsulate cells and/or other biologically relevant components in
microtissues or microgels. Cells or components are uniformly
dispersed within prepolymerized hydrogel using "on-chip"
emulsificaition, for example, using the microencapsulation device
described herein. In preferred embodiments, cells are injected
through a first inlet and prepolymer solution through a second
inlet. Cells and prepolymer solution are allowed to come into
contact (combined aqueous phase) and flow into a droplet generating
nozzle. The combined aqueous phase is allowed to come into contact
with the oil (continuous oil phase.) Speed of flow (flow rate)
and/or nozzle size and/or nozzle shape control droplet size.
Droplets pass through a mixing region of the channel within the
device to disperse cells within prepolymer. Droplets then pass, in
preferred embodiments, through a UV irradiation region of the
channel or device (e.g., a portion of the device in contact with a
UV source, e.g., a curing lamp. Microtis sue size is controlled by
the relative flow rates of the combined aqueous phase (QP) and the
continuous oil phase (QO), and increases with prepolymer:oil flow
ratio. Microtissues are collected from an outlet of the device.
[0058] This technique is referred to herein as "on-chip" emulsion
or "on-chip" emulsification.
[0059] In the absence of an encapsulation device as described
above, photo masking techniques can be used to generate microscale
patterns, e.g., exposed substrate patterned in miscoscale spots
using photomasking techniques. The skilled artisan, however, will
appreciate the enhanced efficiency of the microencapsulation device
technology.
[0060] The above described technology can likewise be used to
generate microgels, by replacing cells with other biologically
relevant compounds, e.g., ECM proteins, etc.
[0061] A preferred process features the use of a photopolymerizable
hydrogel, e.g., a photopolymerizable PEG-based hydrogel. Other
suitable hydrogels for use in the invention include, but are not
limited to, alginates and the like. While these hydrogels are not
photopolymerizable, they are amenable to chemical modification and
can be cross-linked via alternative means (e.g., ionic croslinking)
in an emulsion. Preferably, hydrogels are biocompatible.
[0062] In exemplary embodiments, the hydrogel is functionalized for
cell support and/or assembly purposes. Such scaffolds are also
referred to as chemically modulatable scaffolds. For example,
scaffolds can be modulated to include streptavidin, biotin, DNA,
ECM peptides (e.g., RGD peptides) and the like, also referred to
herein as "affinity biomolecules." In certain embodiments,
conjugation of an "affinity biomolecule" is accomplished via
incorporation of a modified polymer chain, e.g., an acrylate
modified or conjugated chain. Preferably, affinity biomolecules or
other functional groups for later attachment of biomolecules are
incorporated into polymeric hydrogels in a manner such that they
are accessible on the surface of microgels and/or micro tissues of
the invention.
[0063] In some embodiments, cells and prepolymer can be mixed off
chip, for example, where cells and prepolymer are compatible, e.g.,
cells (or biomolecules) are not adversely affected by longer
association with prepolymer. In such embodiments, only a single
aqueous stream is caused to contact the oil stream.
[0064] In certain embodiments, the microgels or microtis sues are
engineered to include one or more adherence materials to facilitate
maintenance of the desired phenotype of the encapsulated cells. The
term "adherence material" is a material incorporated into a
construct of the invention to which a cell or microorganism has
some affinity, such as a binding agent. The material can be
incorporated, for example, into a hydrogel prior to seeding with
parenchymal and/or non-parenchymal cells. The material and a cell
or microorganism interact through any means including, for example,
electrostatic or hydrophobic interactions, covalent binding or
ionic attachment. The material may include, but is not limited to,
antibodies, proteins, peptides, nucleic acids, peptide aptamers,
nucleic acid aptamers, sugars, proteoglycans, or cellular
receptors.
[0065] The type of adherence material(s) (e.g., ECM materials,
sugars, proteoglycans etc.) will be determined, in part, by the
cell type or types to be cultured. ECM molecules found in the
parenchymal cell's native microenvironment are useful in
maintaining the function of both primary cells and precursor cells
and/or cell lines. Exemplary ECM molecules include, but are not
limited to collagen I, collagen III, collagen IV, laminin, and
fibronectin.
[0066] Preferred microtissues are on the order of 50 to 250 .mu.m
and comprise about 5-10 cells per microtis sues. Viability of cells
throughout microtissues can be monitored via conventional means.
The microtis sues of the invention have been demonstrated to have
good viability and a relatively good distribution of cells
throughout a population of particles.
[0067] Certain cells, e.g., tumor cells, can proliferate within
microtissues of the invention, as well, providing additional
functionality of the microtis sues of the invention.
IV. Methods of Assembling Microtissues or Microgels.
[0068] Microtissues and/or microgels of the invention are readily
assembled into higher ordered structures, for example, structures
mimicking in vivo tissue architecture. In exemplary embodiments,
microtissues and/or microgels are engineered to include an encoding
biomolecule or affinity ligand and substrates, e.g., cell or tissue
culture substrates, are patterned with a templating biomolecule or
affinity ligand (e.g., a binding partner of the encoding
biomolecule or affinity ligand.) The encoding biomolecule tells the
microtissues and/or microgels what specific templating biomolecule
or ligand to look for. The templating biomolecule or ligand
attracts the complementary microtissues and/or microgels having
encoding biomolecule or ligand within or on the surface. In this
manner, one can make organized tissues, i.e., can organize
microtissues and/or microgels in a pre-specified pattern.
[0069] In some embodiments, further 3D structure is obtainable, for
example, by layering additional cells, for example of a different
cell type, on top of already patterned microtissues and/or
microgels. Moreover, even when patterned in an essentially 2D
pattern, e.g., on a surface, cells within microtissues of the
invention behave as if patterned in 3D, i.e., the cells behave as
if having a 3D architecture based on the microtis sue
structure.
[0070] The Examples herein demonstrate the use of DNA as an
encoding and/or templating biomolecule. Other means of patterning
are envisioned, e.g., biotin or biotin conjugated biomolecules and
complementary streptavidin or streptavidin-conjugated biomolecules.
Cells themselves can also be used to attract certain microtis sues
and/or microgels of the invention in the assembly process. For
example, cell attachment peptides (e.g., RGD) in one population of
microtissues and/or microgels can attract cells and said cells can
attract and bind other microtissues and/or microgels comprising
cell attachment peptides.
[0071] An advantage of the technology of the instant invention is
that any particular cell type can be optimized for function in a
separate population of microtissues prior to assembly into higher
ordered structures. Cells needing longer time in culture, specific
nutrients, etc. can be maintained in one population of microtis
sues and cells having distinct requirements in a second population
of microtis sues. Subsequent assembly enjoys the advantage of each
cell type being in optimal condition when patterned.
[0072] Assembly can also be via physical means. For instance,
microtissues and/or microgels of the invention can be physically
trapped or contained in a vessel or structure, e.g., on a chip,
slide, microchamber, etc. In a preferred embodiment, microtissues
are contained within a microfluidic chip or device. Width of
channels within the chip or device can be altered to control
assembly. A filter can be included at one or more outlets of the
microfluidic chamber to retain microtissues and/or microgels. Wider
channels can accommodate the flow of microtis sues and/or microgels
into chips or devices. Channels can also accommodate media or other
aqueous flow around microtissues and/or microgels. In exemplary
embodiments, microtis sues containing hepatocytes are assembled in
microfluidic chips or devices. Aqueous solutions or media can
include test compounds, e.g., test drugs, etc. Metabolism of such
compounds can further be tested, e.g., in eluate from the
microfluidic chip or device.
[0073] Cells within microtissues, e.g., hepatic cells, can
metabolize compounds in the media or respond to compounds in the
media (media or solution.) In further embodiments, cells from
higher ordered structures can be samples or "biopsied" post
treatment with compound to determine the effect of said compound on
the tissue.
[0074] Cells within microtis sues, e.g., tumor cells can be tested
for response to compounds, e.g., tested for their proliferative
response to compounds. Microtissue sorting can be utilized to
determine profiferative response, or inhibition of proliferation,
as described herein.
[0075] Trans-well-like devices are also envisioned for use in the
instant invention to assemble microtissues and/or microgels.
Trans-wells are devices designed to be inserted into cell culture
ware, e.g., into the wells of culture dishes or multiwell plates.
Trans-well-like devices can have altered membrane properties, e.g.,
larger pore sizes, to facilitate media flow to microtissues and/or
microgels but retain microtissues and/or microgels.
[0076] It is also envisioned that one chip designed to mimic one in
vivo tissue could be integrated with one or more additional chips
mimicking other tissues to generate an "organ on a chip" or
"organism on a chip."
V. Uses
[0077] The methodology and constructs of the invention are useful
in a number of different methods as set forth in more detail
below.
[0078] This platform technology is anticipated to have utility in
the development of engineered tissues for basic research, human
therapies, drug development, drug testing, and disease models. It
is proposed that this technology will be particularly useful for
enabling higher fidelity construction of complex engineered tissue
model systems such as liver and other highly differentiated and
complex tissues, which are notably the tissue-types most relevant
for drug toxicity screening by pharmaceutical companies (e.g., for
testing novel compounds for biocompatibility). Additionally, it is
envisioned that this platform to be useful in basic science
research. Specifically, the engineered constructs can serve as
model systems for studying 3D cell-cell interactions in diverse
fields ranging from stem cell to cancer biology. The rapid and
facile construction of microgels and microtissues, including higher
ordered assemblies of these micro tissues and/or microgels, using
the devices and methodologies described herein enables complex,
organized cellular 3D patterning to be combined with a variety of
screening applications (e.g., genetic model systems, toxicology
screens, etc.). In summary, this technology allows studies of
engineered tissues and cellular model systems of both complexity
and scale that are precluded by current technologies and
methods.
[0079] The methodology of the invention is particularly suited for
use in basic research, modeling disease states, testing potential
drug compounds in vivo, toxicology screening, and the like. In
exemplary embodiments, the invention features constructs in which
cells are patterned to achieve a high degree of similarity to a
chosen in vivo system. For example, constructs featuring
parenchymal cells or highly differentiated cells can be patterned
to mimic the physiologic properties of the corresponding or source
tissue from which the cells are derived. In certain embodiments,
the function of parenchymal or highly differentiated cells is
enhanced in constructs of the invention by choosing particular
combinations of parenchymal and/or non-parenchymal cells, for
example, parenchymal cells and stromal cells (e.g., fibroblasts.)
Combinations of cells can be patterned at distinct locations within
the constructs of the invention and/or within adjacent locations
within the constructs (e.g., to facilitate cell-cell communication
between distinct cell types.) In exemplary embodiments, the
constructs feature cell populations patterned to mimic interactions
between cells involved in the organization of tissues in vivo.
[0080] The methodology of the instant invention provides for the
generation of 3D microtissue-based "miniature" organs on a chip. In
exemplary embodiments, the invention provides for the generation of
3D microtissue-based "miniature" liver on a chip (e.g. for
metabolism, drug response or disease modeling). Primary hepatocytes
are encapsulated in 3D within, for example, PEG material, along
with co-encapsulated cell types or signals needed to stabilize
their function. Miniaturized versions of these 3D gels (FIG. 12)
can be packed into a microfluidic chamber (like a packed bed
reactor) and media can be perfused through the interstitial spaces
between the gels.
[0081] Alternatively, rather than a microfluidic device,
microtissues, e.g., hepatic tissues, can be placed in a
"Transwell"-like chamber where tissues are separated from fluid by
a membrane. Preferably, membrane pores are of a size sufficient to
capture microtissues or microgels but allow for the ready exchange
of media components. Perfusion can then occur through the fluid
chamber and interact with the microtis sues, allowing metabolism
and secreted products to be detected, for example, in the outlet of
an appropriate apparatus (FIG. 13).
[0082] The methodology of the invention is generally applicable to
assembly of any type of microtis sue, whether by DNA or by packing
in a microfluidic chamber. In exemplary aspects of the invention,
the microtissues can be hepatic microtissues, as described above,
or cancer microtissues (see below), etc.
[0083] It has been possible, for example, to leverage the
throughput and modularity of the encapsulation devices of the
invention towards an integrated in vitro 3D tumor model platform.
Existing 3D culture models typically require large cell numbers and
use complex endpoints (e.g. imaging or bulk biochemical assays),
and many offer only limited control of the microenvironment (e.g.
spatial cell density in spheroid cultures). Using a large-particle
flow sort and analyzer, we have demonstrated the ability to define
microtis sue populations by homotypic or heterotypic cell
densities, and rapidly read responses to microenvironmental cues
such as ECM, growth factors, and inhibitors.
[0084] In an exemplary aspect, a in vitro 3D tumor model platform
combines the split-injection encapsulation chip (described in the
Examples) with a large-particle flow analyzer and sorter (COPAS
Biosort by Union Biometrica). Cells can be encapsulated within
microtissues (5000/min) that can contain tumor cells, stromal
cells, and various entrapped ECM. These microtissues can then be
further enriched using the flow sorter that measures the
fluorescence intensity each microtissue.
[0085] The following materials, methods and examples are meant to
be illustrative only and are not intended to be limiting.
Introduction to the Examples
[0086] The three-dimensional microscale architecture of living
tissues provides vital environmental cues, including extracellular
matrix, soluble factors and cell-cell interactions..sup.1-2
Paracrine and autocrine cell signaling are critical factors guiding
tissue development.sup.3-4 and maintenance,.sup.5-6 and
dysregulation of these cues contributes to the pathogenesis of
diseased states such as cancer..sup.7-9 Understanding and emulating
these cell-cell interactions has been shown to be critical in
engineering functional tissues in both 2D.sup.10-13 and
3D.sup.14-16 systems. In 3D culture, top-down approaches for
organizing multiple cell types such as dielectrophoresis,.sup.17-18
photopatterning,.sup.19-20 and microfabrication.sup.21 provide
high-precision control over cell placement, but are challenging to
scale-up for the assembly of mesoscale tissues.
[0087] In contrast, bottom-up methods, wherein small tissue
building blocks are assembled into larger structures, have
potential for constructing multicellular constructs in a facile,
scalable fashion..sup.22-26 Living tissues are comprised of
repeating units on the order of hundreds of microns; therefore,
synthetic microtis sues comprised of cell-laden hydrogels in this
size range.sup.27 represent appropriate fundamental building blocks
of such bottom-up methods. Synthetic microtissues of this size have
been previously assembled in packed-bed reactors.sup.22, 28 or by
hydrophobic/hydrophilic interactions.sup.24, 29 but without the
ability to specify the placement of many different microtissues
relative to one another. One potential method for controlled
assembly of heterostructures would be to incorporate the
specificity of biomolecular interactions with surface templating to
direct assembly. This approach could allow for scalable patterning
of multiple cell types into arbritrary architectures with high
precision.
[0088] In this work, we harness the well-characterized molecular
recognition capabilities of DNA to achieve rapid templated assembly
of multiple microtis sue types (FIG. 1). This method is enabled by
the high-throughput production of spherical cell-laden microtissues
from a microfluidically-derived, monodispersed emulsion of a
photocurable hydrogel. Cell-laden microtissues are derivatized with
single-stranded oligonucleotides and integrated with custom DNA
microarray templates. Orthogonal DNA sequences are used to specify
the assembly of multiple cell types over large (.about.mm) length
scales with high capture efficiency. This fusion of `bottom-up`
(templated assembly) and `top-down` (microfluidics and robotic
spotting) approaches allows for unprecedented control over
mesoscale tissue microarchitecture and exemplifies the potential of
integrating disparate fabrication strategies.
EXAMPLES
[0089] Patterning multiple cell types is a critical step for
engineering functional tissues, but few methods provide
three-dimensional positioning at the cellular length scale. Here,
we present a "bottom-up" approach for fabricating multicellular
tissue constructs that utilizes DNA-templated assembly of 3D
cell-laden hydrogel microtissues. A flow focusing-generated
emulsion of photopolymerizable prepolymer is used to produce 100
.mu.m monodisperse microtissues at rates of 30 Hz (10.sup.5/hr).
Multiple cell types, including suspension and adherently cultured
cells, can be encapsulated into the microtissues with high
viability (.about.97%). We then use a DNA coding scheme to
self-assemble microtissues "bottom-up" from a template that is
defined using "top-down" techniques. The microtissues are
derivatized with single-stranded DNA using a biotin-streptavidin
linkage to the polymer network, and are assembled by
sequence-specific hybridization onto spotted DNA microarrays. Using
orthogonal DNA codes, we achieve multiplexed patterning of multiple
microtissue types with high binding efficiency and >90%
patterning specificity. Finally, we demonstrate the ability to
organize multicomponent constructs composed of epithelial and
mesenchymal microtissues while preserving each cell type in a 3D
microenvironment. The combination of high throughput microtissue
generation with scalable surface-templated assembly offers the
potential to dissect mechanisms of cell-cell interaction in three
dimensions in healthy and diseased states as well as provide a
framework for templated assembly of larger structures for
implantation.
Example 1
Materials and Methods
Device Fabrication
[0090] Microfluidic device masters were fabricated on 4 inch
silicon wafers using standard photolithographic methods, with SU-8
2050 photoresist (Microchem, MA) spin coated at 1200 rpm to create
125 .mu.m tall features. Masters were coated with trichloro
perfluorooctyl silane (Sigma-Aldrich) for 1 hr in a vacuum
dessicator prior to casting polydimethylsiloxane (PDMS, Dow
Corning) devices. Cured devices with inlet holes made by a 20G
dispensing needle (McMaster-Carr) were bonded to glass slides
following air plasma treatment. In order to ensure a hydrophobic
surface for droplet generation, Aquapel (PPG Industries) was
briefly injected into the device and flushed out with nitrogen.
Ligand Conjugation
[0091] Acrylate-PEG-RGDS (SEQ ID NO: 1) peptide was prepared as
previously described..sup.14 To conjugate streptavidin with
acrylate groups, streptavidin was dissolved in 50 mM sodium
bicarbonate (pH 8.5) at 0.8 mg/ml. Amine-reactive acrylate-PEG-SVA
(3.4 kDa, Laysan) was added at a 25:1 molar ratio and allowed to
react with the protein at room temperature for 2 hours. Conjugated
acrylate-PEG-streptavidin was purified from unconjugated PEG by
washing in PBS with a 30,000 MWCO spin filter (Millipore). The
acrylate-PEG-streptavidin conjugate was then reconstituted to 38
.mu.M streptavidin in PBS, sterile filtered, and stored at
-20.degree. C.
Microtissue Polymerization
[0092] Irgacure-2959 initiator (Ciba) was dissolved at 100 mg/ml in
n-vinyl pyrrolidinone accelerator (Sigma-Aldrich) to make
photoinitiator working solution. The basic 2.times. concentrated
prepolymer solution consisted of 20% w/v poly(ethylene glycol)
diacrylate (PEG-DA, 20kDa, Laysan) and 2% v/v of photoinitiator
working solution. Additional prepolymer ingredients included 38
.mu.M of acrylate-PEG-streptavidin conjugate, 10 .mu.M
acrylate-PEG-RGDS (SEQ ID NO: 1), and/or 1% v/v of fluorescent
microspheres (2% solids, Invitrogen) as markers.
[0093] The final 2.times. prepolymer solution was injected into the
microencapsulation device in parallel with, for cell-free microtis
sues, a 1:1 diluting stream of PBS. Syringe pumps were used to
control the flow rates of the aqueous phases and the oil phase,
which consisted of the perfluoro polyether, Fomblin (Y-LVAC, Solvay
Solexis), with 0-2 w/v % Krytox 157 FSH surfactant (DuPont).
Prepolymer droplets were gelled on-chip by exposure to 500
mW/cm.sup.2 of 320-390 nm UV light (Omnicure S1000, Exfo) for an
approximately one second residence time under typical flow
conditions. Cell-free microtissues were collected in handling
buffer (PBS with 0.1% v/v Tween-20), allowed to separate from the
oil phase, and washed on a 70 .mu.m cell strainer to remove
un-polymerized solutes.
Bead Hybridization
[0094] To stain for the surface-availability of ssDNA bound on
microtissues, 1 .mu.m NeutrAvidin biotin-binding beads
(yellow-green, Invitrogen) were coated with the complementary
5'biotin-DNA (IDTDNA). The original suspension of beads (1% solids)
was diluted 1:10 with BlockAid blocking solution (Invitrogen),
sonicated for 5 minutes, and then incubated with a final
concentration of 4 .mu.M 5'biotin-DNA for 1 hour at room
temperature. Beads were then washed three times in PBS by
centrifugation at 2000.times.g. DNA-functionalized microtissues
were incubated overnight on a room-temperature shaker with coated
beads resuspended to 0.1% solids in BlockAid.
Microarray Spotting
[0095] Microarray templates were printed in-house using a
contact-deposition DNA spotter (Cartesian Technologies) with a
946MP10 pin (Arrayit). Complementary pairs of single-stranded
oligonucleotides used to functionalize microtissues and template
their assembly are listed below and consisted of a poly-A linker
followed by a heterogeneous 20 nucleotide sequence. The
20-nucleotide binding region of A and A' are complementary, B and
B', etc. Sequences were modified with 5'-amine groups for
microarray spotting, and 5'-biotin groups for microtissue
functionalization.
TABLE-US-00001 Label Sequence A 5'-AAAAAAAAAAGCCGTCGGTTCAGGTCATA-3'
(SEQ ID NO: 2) A' 5'-AAAAAAAAAAATATGACCTGAACCGACGGC-3' (SEQ ID NO:
3) B 5'-AAAAAAAAAAAGACACGACACACTGGCTTA-3' (SEQ ID NO: 4) B'
5'-AAAAAAAAAATAAGCCAGTGTGTCGTGTCT-3' (SEQ ID NO: 5) C
5'-AAAAAAAAAAGCCTCATTGAATCATGCCTA-3' (SEQ ID NO: 6) C'
5'-AAAAAAAAAATAGGCATGATTCAATGAGGC-3' (SEQ ID NO: 7) D
5'-AAAAAAAAAATAGCGATAGTAGACGAGTGC-3' (SEQ ID NO: 8) D'
5'-AAAAAAAAAAGCACTCGTCTACTATCGCTA-3' (SEQ ID NO: 9)
[0096] 5'-amino oligonucleotides (IDTDNA) for templating were
dissolved in 150 mM phosphate buffer (pH 8.5) at concentrations up
to 250 .mu.M, and spotted on epoxide coated slides (Corning) at 70%
RH. Patterned slides were then incubated for 12 hours in a 75% RH
saturated NaCl chamber, blocked for 30 minutes in 50 mM
ethanolamine in 0.1M Tris with 0.1% w/v SDS (pH 9), and rinsed
thoroughly with deionized water.
DNA-Directed Assembly
[0097] Microtissues containing PEG-streptavidin were incubated with
1 nmol of 5'-biotin oligonucleotides per 10 ul of packed
microtissues for one hour at room temperature or overnight at
4.degree. C. Un-bound oligonucleotides were removed by washing
microtis sues on a 70 .mu.M cell strainer or using 100,000 MWCO
spin filters. Multi-well chambers (ProPlate, Grace Bio-Labs) were
assembled over templating slides, and DNA-functionalized
microtissues were seeded in a concentrated suspension over the
microarray patterns. Microtissues quickly settled into a monolayer,
which was visually confirmed under a microscope. Unbound
microtissues were washed off the template by gently rinsing the
slide with several ml of handling buffer. Capture efficiency was
quantified by measuring the average seeding density of settled
microtis sues in a 4.times. microscope field of view, divided by
the average capture density over replicate spots on a slide.
Percent of maximum packing fraction was calculated as the ratio of
capture density to the theoretical density of close-packed
circles.
Cell Culture
[0098] J2-3T3 fibroblasts were cultured in Dulbecco's Modified
Eagle Medium (DMEM, Invitrogen) with 10% bovine serum (Invitrogen),
10 U/ml penicillin (Invitrogen), and 10 mg/ml streptomycin
(Invitrogen). TK6 lymphoblasts (suspension culture) and A549 lung
adenocarcinoma cells were cultured in RPMI 1640 with L-glutamine
(Invitrogen) and 10% fetal bovine serum (Invitrogen), 10 U/ml
penicillin, and 10 mg/ml streptomycin. All cells were cultured in a
5% CO.sub.2humidified incubator at 37.degree. C.
Cell Encapsulation
[0099] Prior to encapsulation, adherent cells (J2-3T3 and A549)
were detached with 0.25% trypsin-EDTA (Invitrogen). Cell pellets
were resuspended at cell densities between 10.times.10.sup.6
cells/ml and 30.times.10.sup.6 cells/ml in an isopycnic injection
medium consisting of 20% v/v OptiPrep (Sigma-Aldrich) in serum-free
DMEM. Isopycnic cell suspensions were injected into
microencapsulation devices in place of the diluting stream of PBS,
along with 2.times. prepolymer solution. Gelled microtissues were
collected and handled in culture media. To assess cell viability
after 3 hours, microtissues stained with calcein AM (1:200, 1 mg/ml
in DMSO, Invitrogen) and ethidium homodimer (1:400, 1 mg/ml in
DMSO, Invitrogen) for 15 minutes at 37.degree. C. Alternatively,
microtissues for DNA-templated assembly were marked with
CellTracker Green CMFDA (1:200, 5 mg/ml in DMSO, Invitrogen) or
CellTracker Blue CMAC (1:200, 5 mg/ml in DMSO, Invitrogen) for 1
hour at 37.degree. C.
Imaging and Visualization
[0100] Images were acquired with a Nikon Ellipse TE200 inverted
fluorescence microscope, a CoolSnap-HQ Digital CCD Camera, and
MetaMorph Image Analysis Software. NIH software ImageJ was used to
uniformly adjust brightness/contrast, and pseudocolor, merge, and
quantify images. Confocal images were acquired with an Olympus
FV1000 multiphoton microscope and Olympus Fluoview software.
NIS-Elements software was used to pseudocolor and reconstruct
maximum intensity, slice, and volume views.
Example II
High-Throughput Microtissue Fabrication
[0101] One factor restricting the application of bottom-up assembly
to tissue engineering has been the low throughput of typical
microtissue fabrication approaches to date, many of which are batch
processes..sup.22, 27, 30 We first sought to design a microfluidic
chip to rapidly produce uniform microtis sues. Droplets generated
by flow focusing of aqueous/oil phases are monodisperse and
amenable to photopolymerization..sup.31 Thus, we fabricated a
device to shear photopolymerizable poly(ethylene glycol) diacrylate
(PEG) prepolymer containing cells into droplets in oil for
downstream gelation by UV-light (FIG. 2a). Concentrated pre-polymer
was injected into the microencapsulation device as a separate
stream from the cell suspension (PBS for cell-free microtissues),
where the two aqueous streams were designed to meet before reaching
a flow-focusing junction (FIG. 2b). With a 60 .mu.m nozzle, shear
forces were sufficient to disperse the aqueous combination into
droplets that passed through a corrugated serpentine channel.sup.32
to thoroughly mix the cell-prepolymer solution (FIG. 2c). The
droplets were then polymerized by UV irradiation for 1 second
during transport to the outlet. Resulting microtissues were
uniformly spherical and monodisperse (FIG. 2d). We observed that by
adjusting aqueous vs. oil phase flow rates (FIG. 2e) and oil-phase
surfactant concentrations (FIG. 2f), we could finely control
droplet diameter, and hence microtis sue size, between 30-120
.mu.m.
[0102] At a typical prepolymer flow rate of 200 ul/hr, our device
was capable of achieving a production throughput of 6000
microtissues/min (.about.10.sup.5/hr), two orders of magnitude
faster than other continuous systems such as stop-flow
lithography.sup.33 (.about.10.sup.3 particles/hr) or batch
fabrication processes..sup.27 Microtis sue fabrication by
microfluidic droplet photopolymerization provides precise control
over microtis sue shape and size, whereas photolithographic.sup.27
and molding.sup.22, 24 techniques do not produce spherical gels and
can suffer from resolution limits. Planar microtissue surfaces tend
to adhere non-specifically to hydrophilic surfaces due to the high
water content (>90%.sup.34) of the hydrogel material, whereas
the low contact area of spherical microtissues reduces capillary
adhesion during both handling and assembly. Droplet-based gels have
previously been made using agarose.sup.35 or alginate;.sup.36 here,
we chose a PEG hydrogel material for its biocompatibility and
biochemical versatility. PEG-diacrylate hydrogels have high water
content, are non-immunogenic and resistant to protein adsorption,
and can be easily customized with degradable linkages, adhesive
ligands, and other biologically or chemically active
factors..sup.37
Example III
Microtissue Functionalization With Surface-Encoding DNA
[0103] Having established a method to uniformly produce
microtissues, we next sought to modify our microtissues with
streptavidin for binding biotinylated DNA. To accomplish this,
streptavidin was incubated with amine-reactive acrylate-PEG-SVA
(3.4 kDa). Following purification, the acrylate-decorated
streptavidin was then mixed into the prepolymer and covalently
bound into the acrylate-PEG-acrylate hydrogel network during
gelation by acrylate polymerization (FIG. 3a). Cell-free PEG-SA
microtissues containing conjugated acrylate-PEG-streptavidin were
stained to verify biotin-binding capacity using
biotin-4-fluorescein.We also confirmed the surface-availability of
streptavidin with an anti-streptavidin antibody, which was size
restricted to only the surface of the microtissue (.about.7 nm mesh
size.sup.34). Both biotin fluorescence and antibody staining
intensities increased with the volumetric concentration of
conjugated streptavidin (FIG. 3b).
[0104] With streptavidin incorporated into the hydrogel network, we
were able to encode the microtissues post-polymerization with
5'-biotin terminated oligonucleotides (FIG. 3c).
Streptavidin-biotin based DNA-functionalization of microtis sues is
simple, modular, and cytocompatible. Post-polymerization encoding
of microtissues with biotin-DNA avoids UV damage that would occur
by pre-mixing acrylated-DNA into the prepolymer,.sup.38-39 and
allows the same batch of microtissues to be labeled after culture
in various conditions. Other bioconjugation methods exist to modify
hydrogel networks post-encapsulation, such as maleimide or NHS
chemistries.sup.40 but often require reaction conditions that are
incompatible with maintaining the viability of encapsulated cells.
To ensure that DNA bound to microtissues using the
streptavidin-biotin interaction was available to hybridize with DNA
displayed on a surface, we incubated DNA-encoded microtissues with
1 .mu.m polystyrene beads coated with the complementary
oligonucleotide (FIG. 3c). After washing to remove non-specifically
bound material, microtissues encoded with the complementary
sequence were thoroughly coated with beads visible as bright,
punctuate spots (FIG. 3d). Conversely, beads did not specifically
hybridize to control microtissues (FIG. 3d). In order to maximize
bead-microtissue hybridization, we investigated conjugating
acrylate-PEG-SVA to streptavidin at several molar ratios (FIG. S6).
As expected, microtissues incorporating streptavidin with few
acrylate pendants (10:1 molar ratio, mobility shift assay) did not
promote bead hybridization as effectively as streptavidin modified
with a higher number of acrylate groups (25:1 to 50:1 molar ratio),
which was used for all further studies. Gels incorporating
over-decorated streptavidin (1000:1 molar ratio) were also not as
efficient in mediating bead-microtis sue hybridization, suggesting
that over-modification and/or steric hindrance plays an important
role in DNA-binding capacity.
Example IV
[0105] Binding Efficiency and Specificity of DNA-Templated
Assembly
[0106] Having shown that cell-free microtissues can be coated with
DNA and hybridize specifically to complementary beads, we next
investigated the potential of microtis sue assembly into mesoscale
patterns determined by an encoded template. To create such a
template, we spotted increasing concentrations of DNA (sequence A')
onto a functionalized glass slide using conventional microarray
technology. DNA-functionalized microtissues (A; containing green
marker beads) were allowed to settle onto microarray slides from
suspension, at which time non-hybridized microtis sues were gently
washed off the slide. The number of microtissues bound to
templating array spots increased with higher spotting
concentrations of templating ssDNA (FIG. 4a), plateauing at 250
.mu.M, an order of magnitude higher than typical epoxy-silane based
microarray spotting concentrations. Spots were fully covered by
microtissues at this highest DNA density. To determine the capture
efficiency, we seeded microtissues at varying densities
(microtissues per mm.sup.2, FIG. 4b). At contact-limited
(hexagonally close-packed) seeding concentrations, we achieved 100%
capture efficiency, indicating that if a microtissue settled onto a
complementary spot, hybridization and binding would occur.
[0107] Similar efficiencies have been observed during the
DNA-templated assembly of materials ranging in scale from molecules
to nanoparticles to single cells.sup.23, 41-46. Until now,
DNA-templated assembly has not been extended to larger units such
as microtissues (100 .mu.m), which present unique challenges in
mass transport..sup.47 At these mesoscopic scales, gravity and
friction become important factors in the ability of DNA-coated
surfaces to sufficiently interact. During washing steps, stronger
viscous drag forces on the microtissues necessitate a large number
of hybridization bonds between the microtis sues and templating
surface to overcome microtissue removal. Here, to compensate for
microtissue size, we optimize microtis sue DNA functionalization
and template spotting to achieve high DNA surface densities,
enabling the first demonstration of large structure DNA-templated
assembly.
[0108] During our assembly process, minimal microtissue binding was
observed between spots and on non-complementary templating spots
(FIG. 4b), which was largely made possible by our control over
microtissue shape. This low background binding allowed us to
sequentially pattern multiple microtissue types, each encoded with
an orthogonal oligonucleotide sequence, with over 90% specificity
(FIG. 4c, d) and across large areas in under 15 minutes (FIG. 4e,
f). Furthermore, we were able to build 3D structures (FIG. 4g, h)
by filling template spots (B') with a layer of microtissues (B),
and then seeding a second layer of complementary microtissues (B')
that bind on and around microtissues in the first layer. Together,
these experiments demonstrate the ease of achieving organizational
control at macroscopic length scales by microtissue assembly.
Example V
[0109] DNA-templated assembly of multicellular tissue
constructs
[0110] In order to apply DNA-templated patterning to the assembly
of multicellular constructs, we next focused on encapsulating cells
into uniform and highly viable cell-laden microtis sues. To improve
the consistency of cell encapsulation (FIG. 7), we increased the
specific gravity of our cell suspensions to prevent cell settling
during injection. We chose a density gradient medium (OptiPrep),
based on an iodinated small molecule, that increases specific
gravity without affecting viscosity or cross-linked hydrogel
network density, and easily diffuses out of the polymerized
microtissues. With these changes, we attained cell encapsulation
matching a Poisson distribution (FIG. 5b). In addition, we replaced
the hydrocarbon oil phase with an oxygen-permeable fluorocarbon oil
(Fomblin) to allow immediate quenching of excess free radicals
post-UV exposure..sup.48 Notably, using fluorocarbon oil, cells
were able to tolerate a wide range of total UV exposures
(mJ/cm.sup.2) while maintaining >90% viability (FIG. 5c). As a
result of these changes, several adhesive and suspension cell
lines, including adherent mesenchymal (fibroblasts), nonadherent
mesenchymal (lymphoblasts) and adherent epithelial
(adenocarcinoma), were uniformly encapsulated into microtissues
with consistently high viability (FIG. 5a). Variations in average
viability between cell types (e.g. J2-3T3 vs. TK6) could be due a
number of cell type differences including susceptibility to DNA
damage..sup.49 For cell lines sensitive to UV, photoinitiators in
the visible-light range could be substituted into our material
system..sup.50
[0111] These are many advantages associated with patterning
cellular microtissues rather than single cells..sup.43-44 Firstly,
cells can be encapsulated in a modular scaffold with customized ECM
molecules (e.g. RGDS) (SEQ ID NO: 1) to promote certain phenotypes.
As an example, we added acrylated RGDS (SEQ ID NO: 1) peptide to
the prepolymer during fibroblast encapsulation. By Day 2
post-encapsulation, fibroblasts began spreading within these
adhesive microtissues (FIG. 5d, FIG. 8). Secondly, microtissues
containing one cell type can be first cultured separately to
stabilize homotypic interactions before they are self-assembled
with other microtis sues to activate heterotypic interactions. For
instance, when cultured for several days, adenocarcinoma cells
encapsulated from a single-cell suspension formed multicellular
spheroids (FIG. 5e). In addition, encoding DNA is bound to the
hydrogel scaffold rather than directly onto the cell
membrane,.sup.43-44 where covalently bound ligands may be
susceptible to recycling or may potentially modify cell function.
Encoded microtis sues can remain in assembled patterns for an
extended period of time without additional measures for
immobilization (e.g. embedding in agarose.sup.23), and then removed
for further culture, isolation, and biochemical analysis..sup.27
DNA provides a way for programmed detachment via dehybridization
(e.g. competitive binding with free ssDNA) or cleavage (e.g.
restriction enzymes)..sup.43 Alternatively, patterned microtissues
could be stabilized into a contiguous tissue by a secondary
hydrogel polymerization.sup.29 or cell adhesion between
microtissues to form 3D sheets for implantation (FIG. 9).
[0112] Finally, to demonstrate DNA-templated positioning of
microtis sues containing distinct cell types into pre-defined
patterns, we encapsulated adenocarcinoma cells (blue) and
fibroblasts (green) into separate microtissues and encoded them
with orthogonal DNA sequences (C and D respectively). These
microtissues were then seeded onto an array printed with hexagonal
clusters of complementary DNA (C' centered within 6 spots of D'),
forming co-cultures of the two cell types representative of a tumor
nodule surrounded by stromal cells (FIG. 5f). Multicellular
constructs patterned using this method could be relevant model
systems for studying cancer-stroma interactions in 3D. Notably,
although DNA-templated microtissues are patterned on a 2D template,
cells are encapsulated and respond to a locally 3D
microenvironment, e.g. developing into tumor spheroids (FIG. 5e)
rather than growing as a 2D monolayer..sup.16 Heterotypic signaling
from stromal cells has been shown to contribute to tumor invasion
and metastasis..sup.9 The combination of precise spatial control,
similar to that achieved in 2D,.sup.10 but with a 3D environment,
will be critical toward elucidating such cell signaling
mechanisms.
[0113] Examples I-V present a method to organize multiple cell
types within a 3D microenvironment that integrates the top-down
patterning of a DNA microarray template with the bottom-up assembly
of DNA-encoded, cell-laden microtis sues. This is the first
demonstration of microtissue assembly that is directed by specific
biomolecular interactions. The speed and scalability of the
assembly process is compatible with DNA templates that can be
fabricated by other top-down techniques, such as microfabrication
and micro-contact printing, for a diverse range of features and
patterning resolution. The programmable molecular interaction of
DNA to direct assembly has the potential to be extended to even
larger sets of encoding sequences to create more complex
heterogeneous structures. The ability to precisely control
cell-cell interactions (e.g. cancer-stromal cell,
hepatic-nonparenchymal cell) via microfluidic cell encapsulation
and DNA-templated microtissue assembly provides a unique
opportunity to increase our fundamental understanding of complex
diseases or to construct highly functional tissue-engineered
implants.
Example VI
[0114] This Example demonstrates that it is possible to maintain
the human hepatic phenotype in 3D engineered liver tissues and
demonstrated robust hepatic functions over weeks of culture,
including protein synthesis, human drug metabolism, drug-drug
interaction, and drug-induced liver injury (FIG. 10). Interactions
with fibroblast and endothelial nonparenchymal cells were critical
for the maintenance of hepatic viability and function in 3D
culture; the ligation of .alpha.5.beta.1 integrin via RGDS further
improved hepatic functions (FIG. 10A). More recently, it has been
possible to extend the function of 3D engineered liver tissues even
further, by addition of a micropatterning step pre-encapsulation
(FIG. 11).
[0115] Characterization of hepatic tissues for the expression and
function of human drug-metabolizing enzymes (FIG. 10B) by a
high-throughput Luminex bead PCR assay demonstrated that the
majority (68 of 82) of human drug metabolism-encoding transcripts
were expressed in 3D hepatic tissues (FIG. 10B). Functional
validation of gene expression by treatment with compounds known to
induce or inhibit CYP450 enzymes showed that engineered tissues
accurately predict clinical drug metabolism and interactions (FIG.
10C).
Example VII
[0116] This Example demonstrates the generation of a 3D
microtissue-based liver on a chip (e.g. for metabolism, drug
response or disease modeling). Primary hepatocytes are encapsulated
in 3D within PEG material, along with co-encapsulated cell types
and/or signals needed to stabilize their function. Miniaturized
versions of these 3D gels (FIG. 11) are packed into a microfluidic
chamber (similar to a packed bed reactor) and media is perfused
through the interstitial spaces between the gels. Alternatively,
rather than a microfluidic device, hepatic tissues is placed in a
"Transwell"-like chamber where tissues are separated from fluid by
a membrane. Perfusion then occurs through the fluid chamber and
interacts with the microtissues, allowing metabolism and secreted
products to be detected in the outlet (FIG. 12).
Example VIII
[0117] This Example demonstrates that it is possible to leverage
the throughput and modularity of encapsulation device of the
invention towards an integrated in vitro 3D tumor model platform.
Existing 3D culture models typically require large cell numbers and
use complex endpoints (e.g. imaging or bulk biochemical assays),
and many offer only limited control of the microenvironment (e.g.
spatial cell density in spheroid cultures). Using a large-particle
flow sort and analyzer, we have demonstrated the ability to define
microtissue populations by homotypic or heterotypic cell densities,
and rapidly read responses to microenvironmental cues such as ECM,
growth factors, and inhibitors.
[0118] The in vitro 3D tumor model platform combines the
split-injection encapsulation chip described in detail above with a
large-particle flow analyzer and sorter (COPAS Biosort by Union
Biometrica). Cells are first encapsulated within microtissues
(5000/min) that can contain tumor cells, stromal cells, and various
entrapped ECM. These microtissues are then further enriched using
the flow sorter that measures the fluorescence intensity each
microtissue, correlating with the density of zs-green tumor cells
in each microtissue. (FIG. 15a), and can sort the microtissues
(.about.25 selected/min) into low, medium, and high homotypic cell
density bins (FIG. 15b). Microtissues containing a second labeled
cell type (e.g. fibroblasts labeled DDAO-far red) can also be
sorted by heterotypic density and/or ratio (FIG. 15c). Selected
populations of microtissues are then exposed to stimuli in culture
such as growth factors and inhibitors, and their proliferation over
days is detected on a population level (FIG. 15b).
[0119] Using this platform, we modulated the 3D proliferation of
encapsulated tumor cells by incorporating extracellular (ECM)
proteins (fibronectin promoted growth) and exogenous growth factors
(TGF-.beta. slows proliferation by .about.80%) (FIG. 16). Screening
of several small-molecule inhibitors involved in the TGF-.beta.
pathway identified a molecule that inhibited tumor cell growth on a
2D microplate but had the opposite effect in 3D (FIG. 17),
demonstrating the importance of a 3D model environment in
predicting drug responses.
Other Embodiments
[0120] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate, and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
[0121] In addition, the contents of all references, patents, and
patent applications cited throughout this application are hereby
incorporated by reference.
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Sequence CWU 1
1
914PRTArtificial SequenceSynthetic peptide 1Arg Gly Asp
Ser1229DNAArtificial SequenceSynthetic oligonucleotide 2aaaaaaaaaa
gccgtcggtt caggtcata 29330DNAArtificial SequenceSynthetic
oligonucleotide 3aaaaaaaaaa atatgacctg aaccgacggc
30430DNAArtificial SequenceSynthetic oligonucleotide 4aaaaaaaaaa
agacacgaca cactggctta 30530DNAArtificial SequenceSynthetic
oligonucleotide 5aaaaaaaaaa taagccagtg tgtcgtgtct
30630DNAArtificial SequenceSynthetic oligonucleotide 6aaaaaaaaaa
gcctcattga atcatgccta 30730DNAArtificial SequenceSynthetic
oligonucleotide 7aaaaaaaaaa taggcatgat tcaatgaggc
30830DNAArtificial SequenceSynthetic oligonucleotide 8aaaaaaaaaa
tagcgatagt agacgagtgc 30930DNAArtificial SequenceSynthetic
oligonucleotide 9aaaaaaaaaa gcactcgtct actatcgcta 30
* * * * *