U.S. patent application number 16/234668 was filed with the patent office on 2019-05-09 for engineered lumenized vascular networks and support matrix.
This patent application is currently assigned to Indiana University Research and Technology Corporation. The applicant listed for this patent is Indiana University Research and Technology Corpora. Invention is credited to Abdelkrim Alileche, Dragos Amarie, James A. Glazier, Abbas Shirinifard.
Application Number | 20190136169 16/234668 |
Document ID | / |
Family ID | 44649555 |
Filed Date | 2019-05-09 |
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United States Patent
Application |
20190136169 |
Kind Code |
A1 |
Glazier; James A. ; et
al. |
May 9, 2019 |
Engineered Lumenized Vascular Networks and Support Matrix
Abstract
Disclosed herein are capillary fabrication devices comprising
living cells within a support medium. Culture of the cells produces
viable lumenized capillary networks with natural or pre-determined
geometries and ECM and basement membrane associated with the
capillary networks. The capillary networks and the ECM and basement
membrane detachable from the capillary networks are useful for
tissue engineering applications.
Inventors: |
Glazier; James A.;
(Bloomington, IN) ; Alileche; Abdelkrim; (Boise,
ID) ; Shirinifard; Abbas; (Memphis, TN) ;
Amarie; Dragos; (Bloomington, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Indiana University Research and Technology Corpora |
Indianapolis |
IN |
US |
|
|
Assignee: |
Indiana University Research and
Technology Corporation
Indianapolis
IN
|
Family ID: |
44649555 |
Appl. No.: |
16/234668 |
Filed: |
December 28, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15050356 |
Feb 22, 2016 |
10196596 |
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16234668 |
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13635043 |
Sep 14, 2012 |
9267099 |
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PCT/US2011/028492 |
Mar 15, 2011 |
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15050356 |
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61313886 |
Mar 15, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/00 20180101; C12M
23/10 20130101; C12N 5/0068 20130101; A61K 35/44 20130101; C12M
21/08 20130101; C12M 23/20 20130101; C12N 2533/90 20130101 |
International
Class: |
C12M 3/00 20060101
C12M003/00; C12M 1/00 20060101 C12M001/00; C12M 1/22 20060101
C12M001/22; A61K 35/44 20060101 A61K035/44; C12N 5/00 20060101
C12N005/00 |
Claims
1.-29. (canceled)
30. A formed capillary network made using a device, the device
comprising: living cells; a cell-culture surface; and a support
generating medium, wherein a gel forming material is substantially
dissolved in the cell-culture medium and forms a support medium on
the cell-culture surface.
31. The capillary network of claim 30 wherein the capillary network
comprises tubules having a diameter of at least 5 microns.
32.-34. (canceled)
35. The capillary network of claim 31 wherein the capillary network
geometry is pre-patterned.
36. A method of generating a formed or a forming capillary network,
the method comprising the steps of providing living cells;
dissolving a gel-forming material in the liquid cell-culture medium
to form a support-generating medium; forming a support medium on a
cell-culture surface, wherein the support medium is formed by the
interaction of the support-generating medium with the living cells,
or by the interaction of the support-generating medium with the
cell-culture surface; culturing the living cells under conditions
that permit growth, mobility, and differentiation of the living
cells.
37. (canceled)
38. A method of testing a test substance suspected to promote or
inhibit angiogenesis, the method comprising the steps of contacting
the capillary network of claim 36 with the test substance; and
observing whether the compound promotes or inhibits cell
growth.
39. An ECM and basement membrane produced by the living cells of a
device, the device comprising: living cells; a cell-culture
surface; and a support generating medium, wherein a gel forming
material is substantially dissolved in the cell-culture medium and
forms a support medium on the cell-culture surface wherein the ECM
and basement membrane are detached from a forming or formed
capillary network by changing the pH of the cell-culture medium,
changing the cell-culture temperature, contacting the capillary
network and/or the ECM basement membrane with a stream of liquid,
or any combination thereof.
40.-43. (canceled)
44. A method of repairing or regenerating tissue in vivo, the
method comprising the step of contacting a host's tissue with the
formed capillary network of claim 30.
45.-47. (canceled)
Description
FIELD
[0001] This disclosure generally relates to the field of biomedical
tissue engineering. More specifically, the disclosure relates to
devices for the synthesis of capillary networks with natural or
controlled geometries and their associated extracellular matrix and
basement membrane.
BACKGROUND AND SUMMARY
[0002] Under the proper conditions, endothelial cells or their
precursors/progenitor cells, spontaneously assemble into networks
and differentiate to form lumenized connected capillary
networks.
[0003] Both the forming capillary network and the final capillary
network and its associated extracellular matrix (ECM) have numerous
scientific, industrial and medical applications. In vitro
angiogenesis assays employing the forming capillary network (or
capillary fabrication devices) play a crucial role in identifying
factors involved in vascular development. Such assays are used in
drug development as moderate-throughput screens for angiogenesis
promoters and inhibitors related to wound healing, age-related
macular degeneration, diabetes, cancer, and other diseases.
[0004] The completed networks formed by in vitro angiogenesis
devices are also important in tissue engineering, as vascular
replacements for human implantation, while the ECM of the networks
can be employed as a scaffold implanted into patients to promote
the growth of the patient's own vasculature into the pattern
provided by the ECM.
[0005] Drug development and tissue engineering applications both
require rapid creation of viable lumenized capillary networks
having properties as close as possible to in vivo capillary
networks. Existing in vitro angiogenesis techniques are not capable
of rapidly creating lumenized capillary networks with properties
substantially similar to in vivo networks.
[0006] Current methods of tissue engineering are limited to
relatively thin and/or avascular tissues like skin, cartilage, and
bladder, where post-implantation vascularization from the host is
sufficient to meet the implant's demand for oxygen and nutrients.
Vascularization remains a critical obstacle for engineering
thicker, metabolically demanding organs, such as heart and liver.
Thus the survival of tissue-engineered three-dimensional constructs
in vivo depends on providing enough blood supply to the engineered
tissue and the capacity of the engineered microcirculation to
connect with the existing circulation of the recipient.
[0007] Engineering thicker, metabolically demanding organs, such as
heart and liver, requires techniques for manufacturing microvessels
with highly controlled geometries. Existing microcirculation
engineering techniques are not capable of constructing
controlled-geometry microvessels capable of connection to the host
vessels, expansion of vascular volume accompanying growing tissue,
and prevention of excessive vascular regression.
[0008] Current in vitro capillary fabrication devices are either
quasi-two-dimensional or three-dimensional. Quasi-two-dimensional
devices are categorized as either rapid or long-term. Rapid
quasi-two-dimensional devices consist of a layer of endothelial
cells seeded sub-confluently on top of a thick layer of a
basement-membrane gel which is made of a mixture of collagen,
fibrin, or Matrigel.TM. Depending on the components and mechanical
properties of the gel, endothelial cells align to form a one-cell
thick capillary-like pattern within 1 to 3 days. In rapid devices,
the standard Matrigel.TM. capillary fabrication device (FIG. 4) is
widely used, especially in assaying applications to characterize
anti-angiogenic or pro-angiogenic factors. In the standard
quasi-two-dimensional Matrigel.TM. capillary fabrication device,
cells plated on a thick layer (about 0.5 mm) of Matrigel.TM. are
exposed to high levels of soluble and ECM-bound growth factors. A
high concentration of growth factors in Matrigel.TM. (or even
Growth Factor Reduced (GFR) Matrigel.TM.) results in artifacts and
over-stimulation of cells. Further, cell motility is restricted by
adhesion of cells to the solid Matrigel.TM. and resultant
capillaries have endothelial cells that are abnormally elongated
compared to the in vivo morphology of endothelial cells. The
endothelial cells typically die 24 to 48 hours after forming the
networks in rapid quasi-two-dimensional devices (Ranta et al.
(1998), J Cell Physiol. 176(1):92-98; Vailhe et al. (2001) Lab
Invest. 81(4):439-452); thus these devices are not suitable for
applications requiring viable capillary networks. However, the
lumenized capillary networks formed in the devices disclosed herein
remain viable for about 4 weeks.
[0009] Long-term quasi-two-dimensional devices generally consist of
endothelial cells suspended in normal culture medium and conditions
without inclusion of an extracellular matrix substrate. As the
endothelial cells divide, they form a confluent mono-layer, after
which some differentiate spontaneously to form capillary-like
structures on top of a confluent layer of undifferentiated
endothelial cells. Long-term devices require 2 to 8 weeks of cell
culture, making them unsuitable for high-throughput
experiments.
[0010] Applications of quasi-two-dimensional angiogenesis devices
include, for example, studies of the role and synthesis of
extracellular matrix in vascular morphogenesis; studies of the
roles of adhesion molecules; screening of angiostatic molecules;
functional characterization of endothelial cell lines; studies of
proteases; extracellular protein synthesis; vessel maturation; and
studies of the role of glycation products in diabetes.
[0011] Three-dimensional capillary fabrication devices combine
endothelial cells with a three-dimensional gel which the
endothelial cells then invade. Widely-used three-dimensional in
vitro devices include aortic rings in gelified matrices,
endothelial cells seeded inside a gel or sandwiched between two
layers of gel or between a single layer of gel and a cell-culture
surface, and microcarrier beads coated with endothelial cells. The
forming capillaries in three-dimensional angiogenesis devices are
applied to study the effects of cytokines, metalloproteases and the
fibrinolytic pathway during tubulogenesis, endothelial apoptosis,
the importance of the configuration and composition of the
substrate, the role of cell-adhesion molecules, the effect of
hypoxia on endothelial cells, and for screening of angiogenic and
angiostatic molecules.
[0012] Three-dimensional capillary fabrication devices require many
more cells to achieve the cell densities required for fabrication
of connected capillary networks. Cell distribution in
three-dimensional capillary fabrication devices is not uniform
which may cause over-crowded regions and increased cell death. The
transport of oxygen and other nutrients to three-dimensional
matrices via diffusion limits the thickness of the
three-dimensional matrices in those capillary fabrication devices.
The cell resources in capillary fabrication devices are
prohibitively limited and expensive. Since the cells are embedded
in a solidified gel which limits cell motility, the formation of
capillary-like patterns is slow, taking 1 to 8 weeks. Thus
three-dimensional capillary fabrication devices are inefficient and
slow which make them not suitable for industrial, drug-development
and high throughput applications.
[0013] Thus, improvements are needed for in vitro capillary
fabrication devices.
[0014] Techniques for the vascularization of tissue-engineered
constructs can be broadly grouped into in vitro and in vivo
approaches. In vivo approaches rely on vessel ingrowth from host to
the engineered tissue. This ingrowth process is often limited to
tenths of microns per days, meaning that the time needed for
complete vascularization of an implant of several millimeters is in
the order of weeks. Several strategies have been developed for
improving the ingrowth of vessels from host tissue including
scaffold designs, angiogenic-factor delivery and in vivo
pre-vascularization. In vitro approaches include in vitro
pre-vascularization techniques. In vitro approaches do not rely on
ingrowth of host vessels into entire construct. However,
anastomosis of the vessels in the in vitro-pre-vascularized tissues
is not as fast as in vivo pre-vascularization. In
vitro-pre-vascularization also needs to have proper organization
and geometry to be able to provide enough blood perfusion after
implantation of the engineered tissue.
[0015] The capillary fabrication devices disclosed herein can
produce highly controlled functional lumenized capillary networks
that enhance blood perfusion in the engineered tissue.
[0016] Disclosed herein are capillary fabrication devices for
manufacturing forming capillary networks and formed capillary
networks with natural or controlled geometries the engineered ECM
and basement membrane associated with the capillary networks, and
application of the capillary networks and ECM and basement membrane
for construction of engineered tissues.
[0017] The capillary fabrication devices described herein produce
capillaries which improve in a number of ways on the standard
quasi-two-dimensional Matrigel.TM. capillary fabrication device.
Unlike the standard quasi-two-dimensional Matrigel.TM. capillary
fabrication device, which produces a capillary-like structure
composed of unnaturally elongated endothelial cells, the morphology
of endothelial cells incorporated in the capillary cords and
resulting lumens in the capillary fabrication device described
herein are very similar to those in capillaries formed in vivo,
e.g., in zebrafish embryos and chick allantois.
[0018] It has also been found that the standard
quasi-two-dimensional Matrigel.TM. capillary fabrication device
(even when growth-factor reduced Matrigel.TM. is used) produces a
honeycomb-like network, in which tubules are often composed of a
single endothelial cell stretched and connected to aggregates of
endothelial cells. Most capillary tubules formed in the capillary
fabrication devices described herein are composed of several cell
lengths, and are thus capable of producing mean tubule lengths
comparable to capillaries in vivo (FIG. 1). Capillary networks
produced using the standard quasi-two-dimensional Matrigel.TM.
capillary fabrication device are not viable for more than 48 hours.
The lumenized capillary networks of the capillary fabrication
devices described herein may be viable for up to 4 weeks after
formation of the network.
[0019] The standard quasi-two-dimensional Matrigel.TM. capillary
fabrication device is unable to recapitulate normal cell motility
and proliferation since endothelial cells plated on top of a thick
layer of Matrigel.TM. show no or little proliferation and motility.
The cells do not have properties that are substantially similar to
in vivo cells. For example, mean tubule diameter which has
significant biological importance, is usually less than 5 microns
in the standard quasi-two-dimensional Matrigel.TM. capillary
fabrication device. Formation of narrow tubes (inner diameter <4
microns) in the standard quasi-two-dimensional Matrigel.TM.
capillary fabrication device also indicates high levels of
mechanical stress in the individual cells. Tubes less than 4
microns in diameter do not support natural blood flow. Thus they
are non-functional. However, the diameter of engineered lumenized
capillaries produced using the capillary fabrication devices
described herein were found to range from 5 to 20 microns, which
matches the capillary diameters of zebrafish embryos, chick
allantois and many human tissues.
[0020] In the capillary fabrication devices described herein, only
endothelial lineage cells incorporated in capillary cords are not
motile and show reduced proliferation potency because of strong
junctional complexes; the rest of the cells proliferate and are
motile. Thus, the capillary fabrication devices described herein
are less sensitive to the seeded cell density than the standard
quasi-two-dimensional Matrigel.TM. capillary fabrication device.
This allows formation of capillary networks from limited numbers of
stem cells or from an autograft. An autograft capillary network has
a lower chance of rejection in tissue repair and engineering
applications.
[0021] In the capillary fabrication devices described herein,
similar to long-term in vitro devices, the lumenized capillary
networks remain viable for at least one week after tubulogenesis,
and often remain viable for four weeks or more. In contrast, in
most in vitro capillary fabrication devices, the capillary-like
networks degrade and disappear after tubulogenesis. The longer
viability of the engineered capillary networks described herein
allows for assays to study the effects of pro/anti-angiogenic
factors on both established lumenized capillary networks and the
initial stages of tubulogenesis. The steps of endothelial-cell
tubulogenesis can be recapitulated in quasi-in vivo conditions.
[0022] Use of cell-culture-treated dishes, rather than non-treated
dishes, does not produce a capillary-like pattern, but rather
increases proliferation of seeded endothelial resulting in a
confluent layer of undifferentiated endothelial cells. Typically,
normally adherent cells cultured in non-treated polystyrene dishes
without coating undergo anoikis/apoptosis after a few hours and
die. However, in the capillary fabrication device disclosed herein,
the use of a support-generating medium to form the support medium
on a non-treated polystyrene surface as a cell-culture surface
allows the cells to survive and form capillary networks. Without
being bound by theory, it is believed that the binding of specific
integrin receptors in the endothelial cells contribute to the
initiation of transcription of anti-apoptotic genes,
differentiation and tubulogenesis.
[0023] Current methods of tissue engineering are limited by the
difficulties of forming functional vascular networks in the
engineered tissues. Engineered blood vessels using prosthetic
conduits are not suitable for vessel diameters of less than 6 mm
due to formation of thromboses. Engineered blood vessels which have
a lining of endothelial cells, using cell-sheet engineering or
bioprinting, are limited to large blood vessels. Thus existing
tissue engineering methods are unable to produce functional
vascular networks for tissue engineering. The capillary fabrication
device and custom-patterned capillary fabrication device are
capable of producing functional capillaries which can be integrated
with larger vessels in the host tissue or engineered tissue to form
functional vascular networks. Engineering thicker metabolically
demanding organs, such as heart and liver requires techniques for
manufacturing microvessels with highly controlled geometries.
Existing microcirculation engineering techniques are not capable of
constructing controlled-geometry microvessels capable of connecting
to the host vessels, expansion of vascular volume accompanying
growing tissue, and prevention of excessive vascular regression.
The custom-patterned/controlled-geometry capillary device is
capable of producing functional lumenized capillary networks which
can highly optimize blood perfusion in the engineered tissue.
[0024] In medically-oriented tissue engineering applications
autologous resources of cells are limited. Thus engineering tissues
from autologous components is often not practical. Extracellular
components are highly conserved and known to enhance and regulate
growth/regrowth and differentiation of cells in engineered tissues.
Use of organically-fabricated ECM and basement membrane (formed
using the devices disclosed herein) of non-autologous or non-human
(like mouse) origin can be useful for medical tissue-repair and
tissue engineering applications with minimal transfer of external
factors and thus significant reduction of the likelihood of
rejection after implant.
[0025] The following various embodiments are provided:
[0026] 1) A device for fabrication of engineered capillary networks
comprising:
[0027] living cells;
[0028] a cell-culture surface; and
[0029] a support-generating medium, the support-generating medium
comprising a gel forming material and a liquid cell-culture medium,
wherein the gel forming material is substantially dissolved in the
cell-culture medium and forms a support medium on the cell-culture
surface.
[0030] 2) A custom-patterned capillary fabrication device
comprising:
[0031] living cells;
[0032] a cell-culture surface; and
[0033] a support-generating medium, the support-generating medium
comprising a gel forming material and a liquid cell-culture medium,
wherein the gel forming material is substantially dissolved in the
cell-culture medium and forms a support medium on the cell-culture
surface, and wherein the cell-culture surface comprises a
network-like pattern containing regions of varying
hydrophobicity.
[0034] 3) The device of any of clauses 1 to 2 wherein the cells are
of human origin.
[0035] 4) The device of any of clauses 1 to 3 wherein the living
cells are of endothelial lineage.
[0036] 5) The device of any of clauses 1 to 4 wherein the cells are
selected from the group consisting of embryonic stem cells,
endothelial progenitor cells, circulating endothelial cells, and
lymphatic endothelial cells.
[0037] 6) The device of any of clauses 4 to 5 further comprising at
least one additional cell type.
[0038] 7) The device of clause 6 wherein the additional cell type
is selected from the group consisting of pericytes, smooth muscle
cells, fibroblasts, and any combination thereof.
[0039] 8) The device of any of clauses 1 to 7 wherein one or more
cells are modified cells.
[0040] 9) The device of any of clauses 1 to 8, wherein the
cell-culture surface comprises at least one hydrophobic region.
[0041] 10) The device of any of clauses 1 to 9 wherein the
cell-culture surface comprises a coating of at least one
temperature sensitive polymer.
[0042] 11) The device of clause 10 wherein at least one temperature
sensitive polymer is poly(N-isopropylacrylamide).
[0043] 12) The device of any of clauses 1 to 11 wherein the
cell-culture surface is flat.
[0044] 13) The device of clause 12 wherein the cell-culture surface
is selected from the group consisting of petri dishes, well-plates,
slides, and coverslips.
[0045] 14) The device of any of clauses 1 to 13 wherein the
cell-culture surface is comprised of a material selected from the
group consisting of non-treated polystyrene, glass, a temperature
sensitive polymer, and any combination thereof.
[0046] 15) The device of any of clauses 1 to 14 wherein the
cell-culture surface further includes meshes or scaffolds.
[0047] 16) The device of any of clauses 1 to 15 wherein the
cell-culture surface is modified by etching, stamping, contact
printing, UV laser ablation, or any combination thereof.
[0048] 17) The device of any of clauses 1 to 16 wherein the
cell-culture medium is a defined cell-culture medium.
[0049] 18) The device of any of clauses 1 to 16 wherein the
cell-culture medium comprises serum albumin.
[0050] 19) The device of any of clauses 1 to 18 wherein the
cell-culture medium comprises a bicarbonate-base or HEPES
buffer.
[0051] 20) The device of any of clauses 1 to 19 wherein the
gel-forming material comprises at least one extracellular matrix
(ECM) protein.
[0052] 21) The device of any of clauses 1 to 20 wherein the gel
forming material comprises at least one protein selected from the
group consisting of laminin, collagen IV, heparan sulfate
proteoglycans, entactin/nidogen, TGF-.beta., epidermal growth
factor, insulin-like growth factor, fibroblast growth factor, and
tissue plasminogen activator.
[0053] 22) The device of any of clauses 1 to 20 wherein the
gel-forming material is Matrigel.TM..
[0054] 23) The device of any of clauses 1 to 20 wherein the
support-generating medium comprises Matrigel.TM. diluted from 1 to
30 to about 1 to 60 times in the liquid cell-culture medium.
[0055] 24) The device of any of clauses 1 to 20 wherein the
support-generating medium is dissolved in the liquid-cell culture
medium to yield an ECM protein concentration of from about 170
.mu.g ECM proteins per ml of liquid-cell culture medium to about
350 .mu.g ECM proteins per ml of liquid-cell culture medium.
[0056] 25) The device of any of clauses 1 to 24 wherein the support
medium harbors the living cells.
[0057] 26) The device of any of clauses 1 to 25 wherein the support
medium has a thickness of less than 150 microns.
[0058] 27) The device of any of clauses 1 to 25 wherein the support
medium has a thickness of less than 50 microns.
[0059] 28) The device of any of clauses 1 to 25 wherein the support
medium has a thickness from 50 microns to 100 microns.
[0060] 29) The device of any of clauses 1 to 25 wherein the support
medium has a thickness from 20 microns to 40 microns.
[0061] 30) A formed capillary network made using the device
according to any of clauses 1 to 29.
[0062] 31) The capillary network of clause 30 wherein the capillary
network comprises tubules having a diameter of at least 5
microns.
[0063] 32) The capillary network of clause 30 wherein the capillary
network comprises tubules having a diameter of at least 10
microns.
[0064] 33) The capillary network of clause 30 wherein the capillary
network comprises tubules having a diameter of at least 15
microns.
[0065] 34) The capillary network of clause 30 wherein the capillary
network comprises tubules having a diameter of at least 20
microns.
[0066] 35) The capillary network of any of clauses 30 to 34 wherein
the capillary network geometry is pre-patterned.
[0067] 36) Use of the device according to any of clauses 1 to 29 to
generate a formed capillary network.
[0068] 37) Use of the device of any of clauses 1 to 29 to generate
a forming capillary network.
[0069] 38) The use according to any of clauses 36 to 37 wherein the
living cells are contacted by a test substance suspected to promote
or inhibit angiogenesis.
[0070] 39) An ECM and basement membrane produced by the living
cells of the device according to any of clauses 1 to 29, wherein
the ECM and basement membrane are detached from a forming or formed
capillary network.
[0071] 40) Use of the device according to any of clauses 1 to 29 to
generate an ECM and basement membrane that are detachable from a
forming or formed capillary network.
[0072] 41) The use according to clause 40 wherein the ECM and
basement membrane are detached from the capillary network by at
least changing the cell-culture temperature.
[0073] 42) The use according to any of clauses 40 to 41 wherein the
ECM and basement membrane are detached from the capillary network
by at least changing the pH of the cell-culture medium.
[0074] 43) The use according to any of clauses 40 to 42 wherein the
ECM and basement membrane are detached from the capillary network
by at least contacting the capillary network and ECM and basement
membrane with a stream of liquid.
[0075] 44) Use of a capillary network produced by the device of any
of clauses 1 to 29 to repair or regenerate tissue in vivo.
[0076] 45) Use of the ECM and basement membrane of clause 39 in an
in vitro tissue engineering application.
[0077] 46) Use of the ECM and basement membrane of clause 39 in a
cell-sheet engineering application.
[0078] 47) Use of the ECM and basement membrane in a bioprinting
application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0079] FIG. 1 shows cultured human umbilical vein endothelial cells
(HUVECs) after 7 days of culture using the capillary fabrication
device. Calcein AM was loaded for fluorescent imaging at 10 .mu.M.
White arrows show segments of a viable lumenized capillary network.
A typical undifferentiated HUVEC is about 30 .mu.m in vitro. When
integrated into a tubule, a typical HUVEC is about 50 to 60 .mu.m.
The lumenized capillary lengths range 100 .mu.m to 1500 .mu.m.
ECFCs also formed capillaries when cultured using the capillary
fabrication device for at least 4 days (not shown).
[0080] FIG. 2 shows viable lumenized capillary networks formed from
HUVECs after 36 hours in the disclosed capillary fabrication device
as described herein. They remained viable 7 days later. White
arrows show the lumen in the disclosed capillary fabrication device
as described herein. The lumenized capillaries have a diameter of
10 to 15 .mu.m. Arrow heads show endothelial cells which have not
integrated into the lumenized capillary network. A typical
undifferentiated HUVEC is about 30 .mu.m in vitro. When integrated
into a tubule, a typical HUVEC is about 50 to 60 .mu.m. Bar, 50
.mu.m.
[0081] FIG. 3 shows the formation of support medium. After covering
the cell-culture surface with support-generating medium containing
dissolved gel-forming material, the support-generating medium
forms/deposits a loosely-connected support medium. (A), (B), (C),
(D) show phase-contrast images of the support medium at 30 minutes,
3 hours, 6 hours and 12 hours after plating. Support medium is the
darker patches of material (black arrow-heads in (A) and (B)).
Support medium covers the entire surface after about 6 hours (C).
Support medium coverage does not change significantly after 6
hours. (D) The support medium after 12 hours. The gray background
is the hydrophobic cell-culture surface. Bar, 20 .mu.m.
[0082] FIG. 4 shows components of a standard two-dimensional
Matrigel.TM. capillary fabrication device. Matrigel.TM. solidifies
at room temperature. Endothelial cells are plated on top of a layer
(300-500 .mu.m) of solidified Matrigel.TM.. Cells are covered by
about 2 mm of liquid culture medium. A quasi two-dimensional
capillary-like network one cell length thick forms on top of the
solidified Matrigel.TM..
[0083] FIG. 5 shows components of the capillary fabrication device.
Support-generating medium which contains a dissolved gel-forming
material forms a gel of support medium of about 10 to 30 .mu.m in
thickness. Cells are covered by about 2 mm of support-generating
medium. Lumenized capillary networks form inside the support
medium.
[0084] FIG. 6 shows detached ECM generated using a capillary
formation device. The ECM was detached from the bottom of a
polystyrene dish by making the liquid culture media slightly acidic
(pH.about.6.0-6.5). Arrows show manufactured cables. Scale
bar.about.250 .mu.m.
[0085] FIG. 7 shows the formation of capillaries by HUVECs cultured
on a polystyrene dish having regions coated with support medium and
regions coated with highly hydrophobic PDMS. HUVECs grew and formed
capillary networks on the polystyrene coated with support medium
but did not grow and form networks on regions coated with PDMS. The
boundary between PDMS regions and polystyrene (with support medium)
regions are demarcated by two black lines. The black arrow shows a
formed capillary. Scale bar.about.250 .mu.m.
DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS
[0086] For the purposes of promoting an understanding of the
principles of the disclosure, reference will now be made to
illustrative embodiments. It is understood that no limitation to
the scope is intended. It is further understood that any
alterations and modifications to the illustrated embodiments and
further applications of the principles as may occur to one skilled
in the art to which this invention pertains are included within the
scope of the disclosure.
[0087] As used herein, the phrase "capillary fabrication device"
generally refers to a cell-culture which provides an environment
for manufacturing capillary-like patterns.
[0088] As used herein, the phrase "controlled-geometry capillary
fabrication device" or "custom-patterned capillary fabrication
device" generally refers to a cell-culture which provides an
environment for manufacturing capillary-like networks which have a
predefined geometry including tubule lengths, locations and
connectivity.
[0089] As used herein, the phrase "forming capillary network"
generally refers to a group of cells in a capillary fabrication
device which are organizing to form one or more tubules.
[0090] As used herein, the phrase "formed capillary network"
generally refers to a group of cells in a capillary fabrication
device which are organized into one or more tubules.
[0091] As used herein, the phrase "ECM and basement membrane"
generally refers to the extracellular matrix proteins, growth
factors, and other components that are associated with the forming
or formed capillary networks. The ECM and basement membrane may be
produced and/or modified by the cells in the forming or formed
capillary network. In general, ECM includes basement membrane which
is a laminar organization of extracellular proteins (and
associated/other components) including collagen type IV and
laminin.
[0092] As used herein, the phrase "cell-culture surface" generally
refers to a solid which can physically support cells and support
medium, and the resulting capillaries.
[0093] As used herein, the phrase "non-treated polystyrene" refers
to a polystyrene cell culture surface that has not been pre-treated
(i.e., prior to contact with the support-generating medium) with an
adherence-enhancing substance, corona discharge, gas-plasma, or
other like treatments. Likewise, the phrase "cell-culture-treated"
refers to cell-culture surfaces providing an adhesive substrate for
cell attachment and survival.
[0094] As used herein, "patterned surface" refers to a cell-culture
surface which has spatially or temporally varying physical or
chemical properties including but not limited to hydrophobicity and
protein binding affinities. A patterned surface may include glass,
biocompatible and biodegradable materials and polymers including
but not limited to non-treated polystyrene and temperature
sensitive polymers. A patterned surface may include coatings of
proteins, polymers, or other materials. A patterned surface may
also include scaffolds, meshes and other 2-dimensional or
3-dimensional structures.
[0095] As used herein, the phrase "temperature sensitive polymer"
refers to a polymer which changes its hydration state depending on
temperature. A temperature sensitive polymer includes but is not
limited to poly(N-isopropylacrylamide).
[0096] As used herein, the phrase "gel-forming material" refers to
one or more components that are capable of existing in a gelatinous
or high viscosity liquid form. A gel-forming material may include
one or more extracellular matrix (ECM) proteins. Extracellular
matrix proteins include, but are not limited to, laminin, collagen
IV, heparan sulfate proteoglycans, entactin/nidogen, TGF-.beta.,
epidermal growth factor, insulin-like growth factor, fibroblast
growth factor, and tissue plasminogen activator, and the like. A
gel-forming material of extracellular matrix proteins includes but
is not limited to Matrigel.TM..
[0097] As used herein, "Matrigel.TM." refers to a commercially
available gelatinous extracellular protein mixture secreted by
Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Such proteins
include laminin, collagen IV, heparan sulfate proteoglycans,
entactin/nidogen, TGF-.beta., epidermal growth factor, insulin-like
growth factor, fibroblast growth factor, and tissue plasminogen
activator, and additional proteins. Stock Matrigel.TM. commercially
available currently has about 13 mg/ml of protein. Approximately,
more than 80 percent of Matrigel.TM. is ECM proteins. The
concentration of ECM proteins is initially about 10.4 mg/ml in
Matrigel.TM. (.about.80% of 13 mg). Therefore, Matrigel.TM. stock
diluted 1:30 in cell-culture medium (support-generating medium)
contains about 347 .mu.g of ECM proteins per ml, and a solution
diluted 1:60 contains about 173 .mu.g ECM proteins per ml.
[0098] As used herein, "Growth Factor Reduced Matrigel.TM." (GFR
Matrigel.TM.) refers to a commercially available gel that is
purified and characterized to a greater extent than standard
Matrigel.TM.. GFR Matrigel.TM. effectively has lower levels of a
variety of growth factors except for TGF-beta which may be bound to
collagen IV and/or sequestered in a latent form that partitions
with the major components in the purification procedure. The major
components: laminin, collagen IV and entactin are conserved while
the level of heparan sulfate proteoglycan is reduced by about
40-50%.
[0099] As used herein, the phrase "liquid cell-culture medium"
generally refers to a liquid capable of growing cells and
dissolving the gel-forming material. The liquid cell-culture medium
may comprise HEPES, or a bicarbonate-based buffer, or other buffer.
Optionally, it may also comprise serum albumin.
[0100] As used herein, the phrase "support-generating medium"
refers to a mixture of liquid cell-culture medium and a gel-forming
material at low density. The density of the gel-forming material in
the support-generating medium is low enough that a substantial
portion of the support-generating medium remains in liquid form
(i.e. dissolved in the liquid cell-culture medium) throughout the
culture period (FIG. 5).
[0101] As used herein, the phrases "support medium" or "support
matrix" refers to a thin soft gel produced spontaneously by the
support-generating medium or by the interaction of the
support-generating medium with the cultured cells, or by the
interaction of the support-generating medium with the cell-culture
surface (FIG. 3).
[0102] As used herein, the phrase "endothelial lineage" refers to
endothelial cells or precursor cells or their offspring that are
capable of differentiating into an endothelial cell. Cells of
endothelial lineage include, but are not limited to, endothelial
progenitor cells, endothelial colony forming cells, vascular stem
cells, embryonic and adult stem cells, circulating endothelial
progenitor cells, angioblasts, microvasculature, arteries, veins,
and lymphatic endothelial cells of different origin including but
not limited to dermal, bladder, cardiac, and pulmonary.
[0103] As used herein, "viable" capillaries refer to lumenized
capillaries comprising living endothelial cells.
[0104] Described herein are devices for fabrication of engineered
capillary networks. In various embodiments, the devices comprise
living cells, a cell-culture surface, a support-generating medium,
and a liquid cell-culture medium. In one illustrative aspect, a
gel-forming material is substantially dissolved in the liquid
cell-culture medium to form a support-generating medium, which then
forms a support medium on the cell-culture surface. As used herein,
"substantially dissolved" gel-forming substance means that little
or no gel-forming material remains in solid form after the gel
forming-material is mixed with the liquid cell-culture medium. The
support medium is formed by the support-generating medium or by the
interaction of the support-generating medium with the cultured
cells, or by the interaction of the support-generating medium with
the cell-culture surface. The support medium may partially or
completely coat the cell-culture surface. The support medium
harbors the living cells. The support medium is not rigid. The
support medium permits, growth, motility, and differentiation of
living cells. The cells may permeate the support medium as they
grow, differentiate, and produce their own ECM and basement
membrane.
[0105] In one embodiment, the capillary fabrication device has a
cell-culture surface that is custom-patterned. In one illustrative
aspect, the cell-culture surface pattern is a network-like pattern
that contains regions of spatially and/or temporally variable
hydrophobicities. In one illustrative aspect, the cell-culture
surface comprises hydrophobicity gradients. In one illustrative
aspect, the cell-culture surface patterns are linear. In one
illustrative aspect, the cell-culture surface patterns are
nonlinear. In another illustrative aspect, the cell-culture surface
comprises both linear and non-linear patterns.
[0106] The cells of the capillary fabrication device may be of
human or non-human origin. In one embodiment, the cells are of
endothelial lineage. In one embodiment, the cells comprise a
plurality of cells types of endothelial lineage. In another
embodiment, the cells comprise one or more cell types of
endothelial lineage and at least one additional cell type not of
endothelial lineage. In one aspect, the cells of non-endothelial
lineage provide support and/or stabilize the forming or formed
capillary networks. In one illustrative aspect, the additional cell
type is selected from the group consisting of pericytes, smooth
muscle cells, fibroblasts, and any combination thereof.
[0107] In one embodiment, the cells of endothelial lineage or
non-endothelial lineage are modified. As used herein, the term
"modified cells" includes, but is not limited to, genetic
modifications, for example, the insertion, deletion, or change in
the expression of a gene or genes in a cell. The term "modified
cells" also includes cells affected by any chemical, mechanical,
electromagnetic or other modifications made to the culture to
modify the cells. Modifications of cells may be made continuously
or intermittently at any time before culturing the cells in the
capillary fabrication device, and/or during and/or after capillary
network formation.
[0108] In one embodiment, the capillary fabrication device
comprises a cell-culture surface having at least one hydrophobic
region. In one illustrative embodiment, the hydrophobic regions
form a pattern. In one illustrative aspect, the pattern comprises a
hexagonal network of narrow bands of less hydrophilic cell-culture
surface having a width of from about 5 microns to about 20 microns,
from about 5 microns to about 15 microns, from about 5 microns to
about 8 microns, from about 5 microns to about 10 microns, from
about 8 microns to about 15 microns, from about 10 microns to about
15 microns, from about 10 microns to about 20 microns, or from
about 12 microns to about 20 microns; and a length of from about
175 microns to about 300 microns, from about 200 microns to about
300 microns, from about 175 to about 250 microns, from about 200 to
about 250 microns, from about 225 to about 300 microns, or from
about 225 to about 25 microns. These various ranges of width and
length of the bands are also contemplated where the term "about" is
not included. The custom-patterned/controlled-geometry capillaries
form on top of the predefined hexagonal network pattern of less
hydrophilic cell-culture surface.
[0109] In one embodiment, the capillary fabrication devices have a
cell-culture surface that is modified to change its hydrophobicity
in certain pre-defined regions by application of a hydrophobic
substance capable of attracting the cells. Illustratively, the
hydrophobic substance may be applied by etching, stamping, contact
printing, and the like.
[0110] In one embodiment, the cell-culture surface is pre-coated
with a layer of a temperature sensitive polymer. The temperature
sensitive polymer may become significantly dehydrated at about
37.degree. C. and significantly hydrated at temperatures lower than
about 32.degree. C. Illustratively, the polymer coating is more
hydrophobic in its hydrated state than in its dehydrated state.
[0111] In one embodiment, the temperature sensitive coating is
applied to the cell-culture surface in a pre-formed pattern. In
another illustrative embodiment, the temperature sensitive coating
is applied to the cell-culture surface uniformly and then UV laser
ablation is used to pattern the coated surface by reducing the
thickness of the coating, thereby reducing the hydrophobicity of
that portion of the cell culture-surface. In one illustrative
aspect, the temperature sensitive polymer is
poly(N-isopropylacrylamide).
[0112] In one embodiment of the capillary fabrication devices, the
cell culture surface is flat. For example, a flat surface may be a
petri dish, well-plate, microscope slide, coverslip, and the like.
In one embodiment, the cell-culture surface is comprised of a
material selected from the group consisting of non-treated
polystyrene, glass, a temperature sensitive polymer, and any
combination thereof. Optionally, the cell-culture surface may
contain meshes or scaffolds to further pattern the capillary
networks in two or three dimensions.
[0113] The capillary fabrication devices may comprise any suitable
liquid medium for cell culture. Several cell-culture media are
known to those of skill in the art. In one embodiment, the
cell-culture medium is a defined medium. In one illustrative
aspect, the cell-culture medium is bicarbonate based. In one
illustrative aspect, the cell-culture medium is HEPES. In one
embodiment, the cell-culture medium contains one or more serum
proteins. In one illustrative aspect, the cell-culture medium
contains serum albumin.
[0114] The gel-forming material of the capillary fabrication
devices is substantially dissolved in the liquid cell-culture
medium to form the support-generating medium. Gel-forming materials
for use in the capillary fabrication devices are known to those of
skill in the art. In one embodiment, the gel-forming material
comprises one or more ECM proteins. Extracellular matrix proteins
include, but are not limited to, laminin, collagen IV, heparan
sulfate proteoglycans, entactin/nidogen, TGF-.beta., epidermal
growth factor, insulin-like growth factor, fibroblast growth
factor, and tissue plasminogen activator, and the like. In one
illustrative embodiment, the gel-forming material of the capillary
fabrication devices is Matrigel.TM..
[0115] In one embodiment, the support-generating medium comprises
Matrigel.TM. diluted from about 20 to about 80 times, from about 20
to about 60 times, from about 20 to about 40 times, from about 30
to about 80 times, from about 30 to about 60 times, from about 30
to about 45 times, or from about 45 to about 60 times in the liquid
cell-culture medium. These various dilutions are also contemplated
where the word "about" is not included.
[0116] In one embodiment, the support-generating medium of the
capillary fabrication devices comprises from about 125 .mu.g to
about 500 .mu.g, from about 125 .mu.g to about 170 .mu.g, from
about 125 .mu.g to about 250 .mu.g, from about 125 .mu.g to about
350 .mu.g, from about 170 .mu.g to about 350 .mu.g, from about 225
.mu.g to about 350 .mu.g, or from about 170 .mu.g to about 225
.mu.g of ECM proteins per ml of liquid cell-culture medium. These
various concentrations are also contemplated where the word "about"
is not included.
[0117] In the capillary fabrication devices described herein, the
support-generating medium forms a support medium after contacting
the cell-culture surface and/or the living cells. In one
embodiment, the living cells permeate the support medium and reside
within the support medium soon after cell plating.
[0118] In one embodiment, the capillary fabrication devices
comprise a support medium that is less than 150 microns thick. In
various other embodiments, the support medium has a thickness of
from about 10 microns to about 150 microns, about 10 microns to
about 125 microns, about 10 microns to about 100 microns, about 10
microns to about 75 microns, about 10 microns to about 60 microns,
about 10 microns to about 50 microns, about 10 microns to about 40
microns, about 20 microns to about 75 microns, about 20 microns to
about 60 microns, about 20 microns to about 50 microns, about 30
microns to about 75 microns, about 30 microns to about 60 microns,
about 30 to about 50 microns, about 40 microns to about 70 microns,
or about 40 microns to about 60 microns. In another embodiment, the
support medium has a thickness of from about 20 microns to about 40
microns. In another embodiment, the support medium has a thickness
of from about 50 microns to about 100 microns. These various
thicknesses are also contemplated where the word "about" is not
included. It is appreciated that the thickness of the support
medium may not be uniform at all portions of the cell-culture
surface.
[0119] In one embodiment, a formed capillary network is described
made using the capillary fabrication devices described herein. In
one illustrative aspect, the formed capillary network comprises
tubules and is substantially lumenized. In one illustrative
embodiment, the capillary network has a pre-patterned geometry. In
one illustrative embodiment, the capillary network comprises
tubules having a diameter of at least 5 microns. In another
illustrative embodiment, the capillary network comprises tubules
having a diameter of at least 10 microns. In one illustrative
embodiment, the capillary network comprises tubules having a
diameter of at least 15 microns. In one illustrative embodiment,
the capillary network comprises tubules having a diameter of at
least 20 microns.
[0120] In various embodiments, the capillary fabrication devices
are used to generate forming or formed capillary networks. In one
embodiment, the capillary fabrication devices are used to assess
the effects of one or more test substances on angiogenesis. In one
illustrative embodiment, the plated cells, or the cells of the
forming or formed capillary network are contacted with a test
substance suspected to promote or inhibit angiogenesis.
[0121] In various embodiments, the ECM and basement membrane
produced by the living cells of the capillary fabrication device
are detached. The ECM and basement membrane may be detached from
the forming or formed capillary networks by various methods. In one
illustrative embodiment, the ECM and basement membrane are detached
from the capillary network by changing the pH of the cell-culture
medium, changing the temperature of the cell-culture medium,
contacting the capillary networks and/or ECM and basement membrane
with a stream of culture medium, or any combination thereof.
[0122] The capillary networks and/or the detached ECM and basement
membrane may be used in in vitro tissue engineering applications,
for example, cell-sheet engineering, bioprinting, and the like.
[0123] The capillary networks and/or detached ECM and basement
membrane may also be used in in vivo tissue engineering
applications, for example, tissue-repair, tissue regeneration, and
the like.
EXAMPLES
Example 1: Sample Capillary Fabrication Devices Using Matrigel.TM.
in the Support-Generating Medium to Culture HUVECs or ECFCs
[0124] Materials.
[0125] Matrigel.TM. is purchased from BD Biosciences. Single-donor
human umbilical cord endothelial cells (HUVECs) are cultured
according to protocols provided by PromoCell.TM.. It is recommended
to use HUVECs passaged less than 4 times. Alternatively,
endothelial colony forming cells (ECFCs) are used. ECFCs may be
extracted from human peripheral and umbilical cord blood.
[0126] A stock of 1.times. Matrigel.TM. is diluted about 30 to
about 60 times with about 4.degree. C. cell-culture medium (from
PromoCell) to produce a homogeneous support-generating medium. The
support-generating medium is plated in non-treated polystyrene
petri dishes as the cell-culture surface which has a hydrophobic
surface. Components of the mixture settle/stick to the bottom of
the dish and form a loosely connected low density environment.
About 3 ml of the support-generating medium is enough for 35 mm
dishes. The support medium thickness formed on the dish is about 20
to about 40 microns or when Matrigel.TM. is diluted about 60 times.
The support medium thickness formed on the dish is about 50 to
about 100 microns or when Matrigel.TM. is diluted about 30 times.
The dishes are incubated for about 30 to about 60 minutes in 5%
CO.sub.2 at 37.degree. C. in a cell-culture incubator. The culture
dishes containing cultured HUVECs and 35 mm dishes of
support-generating medium are transferred from the incubator to the
cell-culture hood at the same time to minimize heat shock.
[0127] After harvesting the cultured HUVECs, the cells are added to
the dishes containing the support-generating medium at about 50 to
about 300 cells/mm.sup.2 to complete the capillary fabrication
device. The capillary fabrication device is then incubated in 5%
CO.sub.2 at 37.degree. C. To produce a fully connected network,
cell densities higher than 150 cells/mm.sup.2 are used. To
distribute the plated HUVECs uniformly, the dishes are gently
swirled alternately clockwise and counterclockwise. The
support-generating medium is exchanged about every 48 hours with
fresh pre-warmed support-generating medium, prepared as above. If
the support-generating medium is removed (for example, after about
60 minutes of incubation at 37.degree. C.), and not replenished
with support-generating medium, then the HUVECs do not form
lumenized capillary networks. After formation of capillary
networks, replacing the support-generating medium with regular
liquid cell-culture medium may keep the capillaries viable, but
formation of new capillaries from cells not integrated in
capillaries, may be significantly reduced.
[0128] Optionally, the suspended cells may be mixed with the
support-generating medium immediately before cell plating (keeping
the dilution ratio of Matrigel.TM. in the support-generating medium
between about 1:30 and about 1:60). This procedure eliminates the
step of incubating the dishes for about 30 to about 60 minutes and
is suited to high-throughput experiments.
[0129] Other sources of modified or unmodified cells may be used,
including those from human and non-human sources. Endothelial
lineage cells may also be used.
[0130] Optionally, the cell culture surface may be a flat surface,
including but not limited to a well-plate, slide, or coverslip.
[0131] A glass culture surface, such as a slide or coverslip,
allows for improved high-resolution imaging of the lumenized
capillary networks using an inverted or upright microscope for
assaying chemical treatments to promote or inhibit vascularization
or other purposes.
[0132] Optionally, other support/stabilizing cells can be added to
the cell culture, including but not limited to pericytes, smooth
muscle cells, fibroblasts, myoblasts or any other cell types
capable of interacting with or stabilizing the forming or formed
lumenized capillary network.
[0133] Optionally, modifications to the cells, support-generating
medium, or cell-culture surface can be made continuously or
intermittently at any time before culturing the cells in the
capillary fabrication device, during culturing in the capillary
fabrication device and during and after capillary formation and
during any subsequent applications. Such modifications are useful
in tissue engineering and tissue repair applications which require
functional lumenized capillary networks.
Example 2: Sample Custom-Patterned/Controlled-Geometry Capillary
Fabrication Devices
[0134] In this example, the cell-culture surface in the capillary
fabrication device has hydrophobicity gradients. For example, the
non-treated polystyrene petri dish in Example 1 can be replaced
with a patterned ultra low adhesive polystyrene dish before plating
support-generating medium. The ultra low adhesive polystyrene dish
provides a cell-culture surface which is more hydrophilic than
regular non-treated polystyrene petri dishes. The pattern on the
ultra low adhesive polystyrene dish comprises a hexagonal network
of narrow bands of 5 to 15 microns in width and 200 to 250 microns
in length of less hydrophilic cell-culture surface. Cells of
endothelial lineage can be used. The
custom-patterned/controlled-geometry capillaries form on top of the
predefined hexagonal network pattern of less hydrophilic
cell-culture surface.
[0135] Optionally, the non-treated polystyrene cell-culture surface
in Example 1 can be pre-coated with a layer of temperature
sensitive polymers, like poly(N-isopropylacrylamide). The
temperature sensitive polymer is dehydrated at 37.degree. C. and
hydrated at temperatures lower than 32.degree. C. The polymer
coating is more hydrophobic in its hydrated state. The coating
hydrophobicity also depends on the thickness of the coating. A
coating thickness of more than about 3 nm results in a hydrophobic
cell-culture surface at 37.degree. C. The non-treated polystyrene
is coated uniformly and then UV laser ablation used to pattern the
coated surface by reducing the thickness of the coating according
to a customized pattern. The cells in the custom-patterned
capillary fabrication device form lumenized capillaries over areas
with thinner coating. The cell-culture surface may also be modified
by methods including, but not limited to, etching, stamping, and
contact printing.
[0136] Use of a temperature sensitive polymer in this protocol
allows detachment of the formed capillary network and the
associated ECM and basement membrane with minimal damage. The
cell-culture temperature can be adjusted to facilitate detachment.
The detached capillary network and the associated ECM and basement
membrane can be used in tissue engineering and tissue repair
applications.
[0137] The custom-patterned capillary fabrication device is capable
of manufacturing capillary networks of specified pattern using
cell-culture surfaces with network patterns consisting of narrow
bands of 5 to 15 microns in width and specified length and
connectivity. The device is capable of producing replicas of
2-dimensional capillary networks like choroidal capillaries in the
retina.
Example 3: Sample Capillary Assay Devices
[0138] Capillary networks are grown as described in Example 1 using
the capillary fabrication device or Example 2 using the
custom-patterned capillary fabrication device combined with one or
modifications with the aim of determining the effect of those
modifications on the cultured cells or on the resulting capillary
networks.
[0139] The capillary assay device can be used for determining the
proangiogenic effects of different isoforms of vascular endothelial
growth factor (VEGF) including soluble and ECM-bound isoforms. The
cells in the capillary assay device are cultured inside the support
medium, thus the device provides quasi-in vivo conditions for the
interaction of VEGF isoforms and the cells. Cells in the
2-dimensional standard Matrigel.TM. capillary fabrication device
are cultured on top of a thick solidified gel which differs
significantly from in vivo conditions.
[0140] The capillary assay device also can be used for determining
the effects of various toxic materials such as, for example, lead,
arsenic, and the like on angionesis. Further, the capillary assay
device may be used to study cellular mechanosignaling related to
angiogenesis, or the pro/anti angiogenic effects of mechanical
stressors such as, for example, ultrasonic waves or altered
pressure. Additionally, the capillary assay device may be used to
study the pro/anti angiogenic effects of magnetic or electric
fields, or electromagnetic waves.
Example 4: Engineered Capillary Networks with Modified Cells
[0141] Cells used in this example can be modified before, during or
after growth of capillary network using the capillary fabrication
device (Example 1) or the custom-patterned capillary fabrication
device (Example 2). After a sufficient period of growth, the
capillaries are detached in a manner causing minimal or no damage
to the capillary network and can be used for in vivo or in vitro
implantation.
[0142] The capillary networks formed using custom-patterned
capillary fabrication device (Example 2) have predefined geometry
which makes them suitable for tissue engineering and repair
applications with specified geometries and metabolic rates.
[0143] In vitro modification of cells used to grow the capillary
networks may be useful in minimizing the chance of rejection in
allotransplantation or xenotransplantation.
Example 5: Engineered ECM and/or Basement Membrane
[0144] Capillary networks are grown as described in Example 1 using
the capillary fabrication device or Example 2 using the
custom-patterned capillary fabrication device. After a sufficient
period of growth, the cells are destroyed, for example, by washing
the capillary network with a liquid, for example, distilled water,
while retaining the ECM and basement membrane. Then the patterned
ECM and basement membrane are detached in a manner causing minimal
or no damage to the capillary network, for example, by using a
stream of culture media, or by using temperature sensitive
polymers. Adjusting the temperature or pH to modify hydrophobicity
can facilitate detachment. For example, adjusting the pH of the
culture media to slightly acidic (pH.about.6-6.5) is found to
facilitate detachment of ECM.
[0145] The thickness of the ECM may be controlled by the
concentration of the support-generating medium. For example, using
diluted Matrigel.TM. as a gel-forming material, the thickness of
manufactured ECM is about 20 to 40 microns when Matrigel.TM. is
diluted at a ratio of 1:60 and about 50 to 100 microns when diluted
at a ratio of 1:30. The ECM has a net-work like morphology. The
support matrix is organized in cables that are about 50 to 500
microns long when cultured for 5 days using a 1:30 dilution of
Matrigel.TM.. The width of the cables is about 15 to 100 microns.
The length of the cables increases over the duration of the culture
period. For example, cables about 1.5 mm long are observed after 10
days of culture. At more dilute concentrations (e.g. Matrigel.TM.
at 1:60) the cables elongate faster. Thus, the dilution of the gel
forming material and duration of culture can be controlled to
generate ECM with customized morphology.
[0146] The cell-remodeled basement membrane and ECM may then be
used to enhance growth/regrowth of vasculature in vivo or in vitro.
In vivo implantation can be examined.
[0147] After in vivo implantation of the cultured ECM and/or
basement membrane, regrowth of capillaries and pericyte recruitment
occurs as cells invade the empty sleeves of basement membrane
and/or ECM left behind by the destroyed capillaries. Since
extracellular components are highly conserved, this
organically-manufactured basement membrane/ECM is useful for
medical tissue-repair applications with minimal transfer of
external factors and thus significant reduction of the likelihood
of rejection after implant.
[0148] The engineered ECM and basement membrane using
custom-patterned capillary fabrication devices (Example 2) have
predefined geometry which makes them suitable for growth/regrowth
of vasculature in tissue engineering and tissue repair applications
with specified geometries and metabolic rates.
Example 6: Use of Engineered ECM and Basement Membrane in
Cell-Sheet Engineering and Bioprinting
[0149] In this example quasi-two-dimensional layers of engineered
ECM and basement membrane (Example 5) are placed in horizontal
layers separated by 100 to 200 micron layers of engineered tissue
in cell-sheet engineering and bioprinting applications. The
resulting enhanced growth of capillary networks in cell-sheet
engineering and bioprinting applications results in a functional
vascular network to provide blood flow to all volumes of the
engineered tissue, enhancing the likelihood of integration of
engineered 3-dimensional tissues.
Example 7: Use of Implantable Capillary Networks in Cell-Sheet
Engineering and Bioprinting
[0150] In this example quasi-two-dimensional layers of formed or
forming capillary networks and associated ECM and basement membrane
(Example 4) formed using the capillary fabrication device or
custom-patterned fabrication device are placed in horizontal layers
separated by 100 to 200 micron layers of engineered tissue in
cell-sheet engineering and bioprinting applications. Integration of
the capillary networks with existing or engineered larger vessels
in these applications results in a functional vascular network to
provide blood flow to all volumes of the engineered tissue,
enhancing the likelihood of integration of engineered 3-dimensional
tissues.
Example 8: Formation and Patterning of Hydrophobic Domains on the
Cell Culture Surface
[0151] Highly hydrophobic domains in the culture surface coating
are generated with polydimethylsiloxane (PDMS) which the cells do
not populate. Moderately hydrophobic domains, which cells invade
and populate, are generated with long carbon chain silanes, for
example trichloro(octyl)silane, trichloro(decyl)silane,
trichloro(dodecyl)silane, trichloro(octadecyl)silane,
trimethoxy(octyl)silane, trimethoxy(decyl)silane,
trimethoxy(dodecyl)silane, trimethoxy(octadecyl)silane,
triethoxy(octyl)silane, triethoxy(decyl)silane,
triethoxy(dodecyl)silane, or triethoxy(octadecyl)silane.
Photoresist masking may be used to generate patterns of moderately
hydrophobic and highly hydrophobic domains on the cell culture
surface.
[0152] For example, a clean glass slide is functionalized with one
or more long carbon silanes (moderately hydrophobic) to create a
carbon chain monolayer. The slide is exposed in a 0.2-2% silane in
organic solvents (e.g. methanol, acetone, toluene, heptane, carbon
tetrachloride, xylene, and the like) followed by a methanol rinse
and then dried under nitrogen gas. The surface is then spin-coated
with a thin, approximately 5-10 .mu.m layer of positive photoresist
(e.g. SPR220, PMMA, and the like). After softbaking the substrate
is exposed to UV light through a mask with a spatial pattern to be
replicated by the cells of the functionalized glass substrates.
After removing the exposed photoresist a thin layer of PDMS is
spin-coated on. The PDMS is then etched by tetrabutylammonium
fluoride (TBAF) solution in tetrahydrofuran (THF) until the layer
becomes 2-5 .mu.m thick. The substrates are then briefly exposed to
UV light without any mask. The photoresist domains masked during
the prior exposure are removed, revealing the silane coated glass
domains surrounded by PDMS domains.
Example 9: HUVECs do not Invade Highly Hydrophobic PDMS Domains
[0153] A capillary fabrication device is utilized to culture HUVECs
as described in Example 1. A PDMS coating is applied as described
in Example 8 to a polystyrene surface. The HUVECs grow and form
capillaries on a polstyrene surface containing a support medium,
but do not grow on regions of the surface coated with PDMS (FIG.
7).
Example 10: Implantation of Viable Capillary Networks
[0154] Capillary networks formed using the capillary fabrication
devices are implanted ex ovo. Chicken eggs are incubated for 3 days
at 37.degree. C. On day 3, eggs are broken and embryos are
transferred to 10 cm dishes. The embryos are grown for 3-4 more
days. On day 6-7, capillary networks formed by HUVECs are implanted
in up to four regions of chorioallantoic membrane (CAM). The CAMs
are scratched with a sharp needle until a small amount of blood
appears. The capillaries are then injected into the CAM using a
micro-pipette.
[0155] The capillary networks are labeled with Calcein AM to
examine their viability. Calcein AM is used as an indicator of
viability because it diffuses out of cells when they die. Implanted
capillary networks are viewed under microscope after implantation.
Viable implanted HUVEC cells are observed 2 days after
implantation.
[0156] While the invention has been illustrated and described in
detail in the foregoing description, such an illustration and
description is to be considered as exemplary and not restrictive in
character, it being understood that only the illustrative
embodiments have been described and that all changes and
modifications that come within the scope of the invention are
desired to be protected. Those of ordinary skill in the art may
readily devise their own implementations that incorporate one or
more of the features described herein, and thus fall within the
scope of the present invention.
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