U.S. patent application number 17/170706 was filed with the patent office on 2021-06-17 for method for engineering three-dimensional synthetic vascular networks through mechanical micromachining and mutable polymer micromolding.
This patent application is currently assigned to CARNEGIE MELLON UNIVERSITY. The applicant listed for this patent is CARNEGIE MELLON UNIVERSITY. Invention is credited to Donna Beer-Stolz, Emrullah Korkmaz, Philip R. LeDuc, O. Burak Ozdoganlar, Yadong Wang, Mary E. Wilson.
Application Number | 20210178636 17/170706 |
Document ID | / |
Family ID | 1000005429482 |
Filed Date | 2021-06-17 |
United States Patent
Application |
20210178636 |
Kind Code |
A1 |
LeDuc; Philip R. ; et
al. |
June 17, 2021 |
Method for Engineering Three-Dimensional Synthetic Vascular
Networks Through Mechanical Micromachining and Mutable Polymer
Micromolding
Abstract
The present invention relates generally to a method that is used
to create three-dimensional synthetic vascular networks.
Micromachining and molding techniques are used to create a template
in a shape that mimics a biological network. Cellular material can
be seeded around the template or a space created by the template
and grown into an engineered tissue-construct.
Inventors: |
LeDuc; Philip R.; (Wexford,
PA) ; Ozdoganlar; O. Burak; (Sewickley, PA) ;
Wilson; Mary E.; (Baltimore, MD) ; Korkmaz;
Emrullah; (Pittsburgh, PA) ; Wang; Yadong;
(Pittsburgh, PA) ; Beer-Stolz; Donna; (Pittsburgh,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CARNEGIE MELLON UNIVERSITY |
Pittsburgh |
PA |
US |
|
|
Assignee: |
CARNEGIE MELLON UNIVERSITY
Pittsburgh
PA
University of Pittsburgh - Of the Commonwealth System of Higher
Education
Pittsburgh
PA
|
Family ID: |
1000005429482 |
Appl. No.: |
17/170706 |
Filed: |
February 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14750620 |
Jun 25, 2015 |
10919183 |
|
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17170706 |
|
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61998388 |
Jun 26, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B29L 2023/00 20130101;
B29L 2031/7534 20130101; B29B 11/14 20130101; B29L 2031/756
20130101; B29C 33/3842 20130101 |
International
Class: |
B29B 11/14 20060101
B29B011/14; B29C 33/38 20060101 B29C033/38 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
FA9550-13-1-0108 awarded by the US Air Force Office of Scientific
Research; W81XWH-11-2-0215 awarded by the US Army/Pittsburgh Tissue
Engineering Initiative; and CMMI0856187 awarded by the National
Science Foundation. The government has certain rights in the
invention.
Claims
1. An artificial vascularized structure comprising: a cavity
embedded in a growth medium, wherein the cavity is formed by the
following process: forming a micromold having a microchannel with a
circular cross-section; flowing a template material into the
microchannel, wherein the micromold has a dissolution rate that
prevents dissolution of the micromold during flowing the template
material; causing the template material to solidify in the
microchannel; dissolving the micromold, leaving a template having a
shape of the microchannel; embedding the template in a growth
medium; solidifying the growth medium; liquefying and then removing
the template material, leaving the cavity in the growth medium in
the shape of the template; and perfusing the cavity with cellular
material.
2. The artificial vascularized structure of claim 1, wherein
forming a micromold comprises: milling a groove on a substrate,
wherein the groove forms a pattern that mimics a biologic
structure; transferring the pattern from the substrate to a first
mold; transferring the pattern from the substrate to a second mold,
wherein the second mold is a mirror image of the first mold; and
joining the first mold and second mold together.
3. The artificial vascularized structure of claim 1, wherein the
cellular material is selected from the group consisting of
endothelial cells and neural cells.
4. The artificial vascularized structure of claim 1, wherein the
growth medium is a collagen gel seeded with a second cellular
material.
5. The artificial vascularized structure of claim 1, wherein the
template material is a gelatin solution.
6. An engineered-tissue scaffold, comprising: a growth medium
having a cavity with an interior surface, wherein the cavity has a
circular cross-section and approximates a shape of a fabricated
vascular-mimetic template, wherein the growth medium is seeded with
a first cellular material, wherein the interior surface of the
cavity is seeded with a second cellular material.
7. The scaffold of claim 6, wherein the growth medium is a collagen
gel.
8. The scaffold of claim 6, wherein the second cellular material is
endothelial cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Nonprovisional
application Ser. No. 14/750,620 filed Jun. 25, 2015, which claims
the benefit under 35 U.S.C. .sctn. 119 of U.S. Provisional
Application Ser. No. 61/998,388, filed Jun. 26, 2014, each of which
is incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0003] The invention relates generally to a method for creating a
vascular structure for use in tissue engineering. More
specifically, the invention relates to a method for fabricating a
machined substrate for creating molds that mimic a natural vascular
structure in size and geometry, the molds being used to create
living cellular structures.
[0004] There is great need for strategies to treat large-scale
tissue loss. From major trauma to disease-related organ failure,
the lives of many could be positively impacted by the ability to
repair or regenerate large-scale tissues and organs. Among military
service members, blast, blunt and penetrating traumas from modern
warfare often results in devastating and complex injuries to organs
and organ systems and frequently lead to mortality. Advanced
regenerative medicine treatment options to heal severely wounded
veterans are of great importance.
[0005] Organ failure contributes to significant morbidity and
mortality in the United States and around the world, contributing
to the health care costs of those affected. Currently, over 110,000
people in the United States are on the organ transplant waiting
list, with kidney and liver being in highest demand. However, there
is a major supply/demand discrepancy as only about 28,000 people
per year receive a life-saving transplant. Unfortunately, a high
percentage of patients will die while awaiting a suitable organ. As
cadaver and living donor sources are inadequate, regenerative
medicine strives to solve the donor organ shortage problem by means
of engineering functional vital organs.
[0006] In addition to clinical application in organ replacement, an
engineered three-dimensional synthetic organ module could also have
implications for use as an in vitro diagnostic assay platform--for
example, in testing of drug and vaccines first-in-human studies.
Current in vitro platforms fail to predict the safety, efficacy and
pharmacokinetics of drugs and vaccines in humans, and animal models
are only slightly better in terms of prognostic ability with
orders-of-magnitude increase in cost. One solution to this problem
is to put the human physiology into an in vitro format that enables
accurate, reproducible, high content diagnostics across the major
organ systems.
[0007] As an evolving interdisciplinary field, tissue engineering
has already produced artificial life-saving tissues. For example,
functional tissue-engineered constructs have been developed and
implanted in vivo to replace tissues such as skin, cartilage, and
bladder. However, these products are not vital organs with high
metabolic demands. Tissue engineering is limited to thin layer
tissues due to a lack of means to vascularize metabolically
demanding cells. As a first step in solving the donor organ
shortage crisis, bioengineered vasculature from the micro- to
macro-scale would provide an enabling technology for vital organ
engineering. Though progress has been made in vascular engineering,
existing approaches to address this challenge remain inadequate. It
would therefore be advantageous to successfully create a complete
three-dimensional microvasculature capable of delivering nutrients
and oxygen while removing wastes for use in many different types of
organs.
BRIEF SUMMARY OF THE INVENTION
[0008] According to embodiments of the present invention is a
method to fabricate vascular-mimetic microfluidic channels using
mechanical micromachining in combination with a polymer molding
process. In one embodiment, a pattern of a vascular structure is
machined into a substrate using a micromilling apparatus. The
substrate is used to create molds of the vascular network. Two
molds, each representing one half of a vascular structure, are
joined together to create an enclosed space having a circular
cross-section. A template of the vascular structure is created by
injecting a fluid into the space and hardening the fluid. The
template is then encased in a growth medium and dissolved or
liquefied, creating a cavity in the medium substantially in the
shape of the vascular network. The cavity is then perfused with
cellular material. Various testing techniques can be used to assess
the viability of the constructs.
[0009] The method of the present invention allows a
three-dimensional vascularized tissue module to be grown in which
capillaries are completely surrounded by parenchymal cells. The
vascularized tissue module serves as a means to enable engineering
three-dimensional tissues with clinically-relevant dimensions. The
method of the present invention, which allows the development of
capillary networks that form close contacts with parenchymal cells,
overcomes limitations of conventional fabrication methods for
creating microchannel features within materials systems. This
method allows one to precisely define and engineer complex vascular
microarchitectures within a cellularized tissue construct through
bottom-up approaches combing three key advantages: (1) mechanical
micromachining to define and create complex vascular-mimetic
architectures; (2) mutable polymer micromolding, including
water-dissolvable and thermally-reversible polymer systems, to
enable precise placement of materials and cells within the defined
microarchitectures; and (3) precision assembly techniques to
facilitate manipulation and alignment of complex microarchitectures
in three-dimensional space.
[0010] This invention represents a significant advance in
microvasculature engineering and impact tissue engineering for many
organs including but not limited to, the liver, kidney, heart,
lung, and brain.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0011] FIG. 1 is a flowchart of the method of the present invention
according to one embodiment.
[0012] FIG. 2 depicts a machined substrate.
[0013] FIG. 3 shows a positive mold cast on the substrate and the
positive mold removed from the substrate.
[0014] FIG. 4 shows a negative mold cast on the positive mold and
the negative mold removed from the positive mold.
[0015] FIG. 5 is an image showing a micromold having a microchannel
formed by joining and aligning two negative molds.
[0016] FIG. 6 is an image showing the circular cross-section of the
microchannel.
[0017] FIG. 7 shows a curved microchannel formed in a mold.
[0018] FIG. 8A shows the creation of a template from the
micromold.
[0019] FIG. 8B illustrates the creation of an example vascularized
co-culture construct from the template.
[0020] FIG. 9 is a series of images showing the distribution of
cells on an interior surface of a microchannel.
[0021] FIG. 10 illustrates HepG2 cells stained with a range of
CellTracker Green BODIPY concentrations: 0.5 .mu.M (A), 1 .mu.M
(B), and 5 .mu.M (C).
[0022] FIG. 11 illustrates the longevity of live cell staining of
HepG2 cells with 5 .mu.M CellTracker Orange prior to cell seeding
in collagen gel.
[0023] FIG. 12 illustrates alternative staining methods tested for
HUVECs: CellTracker Green BODIPY at 1 .mu.M in serum-free media for
45 minutes (A); CellMask Orange at 5 .mu.g/mL in PBS for 10 minutes
(B); and Hoescht 3342 at 2 .mu.g/ml in PBS for 2 minutes (C).
[0024] FIG. 13 illustrates the distribution of endothelial cells
and hepatocytes across the vascular channel diameter (x-axis) by
analysis of confocal microscopy data.
[0025] FIG. 14 illustrates micromolds formed by aligning and
adhering two half-circular molds, optical micrograph demonstrating
the formed circular cross-section at a channel opening, and a
structural design for scalable and stackable three-dimensional
micromolds.
[0026] FIG. 15 illustrates CMC channel sealing using liquid CMC (A)
and water (B) to seal the two CMC slabs.
[0027] FIG. 16 illustrates a precision alignment device to
facilitate alignment and sealing between multiple CMC layers.
[0028] FIG. 17 illustrates the fabrication of a stacked
three-dimensional dissolvable polymer micromold using CMC.
[0029] FIG. 18 illustrates an example of a vascular template made
from a low melting point polyester wax that can maintain a rigid
shape upon demolding, which is well suited for stacked
three-dimensional structures.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Embodiments of the present invention and its advantages are
best understood by referring to the figures. FIG. 1 is a flowchart
showing the steps of the method according to one embodiment. At
step 101, a vascular pattern is machined onto the surface of a
substrate 202. In the preferred embodiment, the substrate 202 is
metal, such as brass or aluminum. The substrate 202 is machined to
create semi-circular patterns that correspond to one-half of a
circular blood vessel. A semi-circular groove 301 machined on the
surface of the substrate 202 can be seen in FIG. 2. As shown in
FIGS. 5 and 6, when matching semi-circular depressions 301 are
aligned, a microchannel 207 having a circular cross-section is
created and mimics the cross-sectional shape of a blood vessel.
[0031] In the preferred embodiment, a high-precision miniature
machine tool 201 with a 160,000 rpm air-turbine, air-bearing
spindle, and micro end mill are used for the machining step.
Miniature cutting tools, having a diameter as small as 10 microns,
can be used. In the preferred embodiment, a four-fluted ball nose
micro-end mill with a 508 micron nominal radius and TiAlN coatings
are used to create the grooves 301 in the substrate 202.
[0032] An end mill with a ball end is used in the preferred
embodiment because it creates a semi-circular depression 301 in the
substrate 202. Unlike three-dimensional lithography methods, for
example, that are limited by a minimum step size in producing
pseudo-curvature, both straight and S-shaped micromilled grooves
301 are continuously curved to form a semi-circular cross-section.
FIG. 7 shows the semi-circular cross-section along an S-shaped
groove 301.
[0033] During milling, feed motions of the substrate 202 can be
controlled using a computer and a three-axis slide. A person having
skill in the art will recognize that various micromilling control
techniques can be used.
[0034] After machining, step 101 includes a two-step reverse
molding process used to transfer the pattern from the substrate 202
to a dissolvable negative mold 204. As a first step of the molding
process, a positive mold 203 is created from the substrate 202,
wherein the mold material fills the grooves 301 in the substrate
202 created during the machining process. The top image of FIG. 3
shows the positive mold 203 formed on the substrate 202 and the
bottom image shows the positive mold 203 removed from the substrate
202.
[0035] Next, a negative mold 204 is created from the positive mold
203. Referring to FIG. 4, the top image shows the negative mold 204
created on the positive mold 203 with the bottom image showing the
negative mold 204 removed from the positive mold 203. In one
respect, the substrate 202 serves as a master mold. However, the
two-step molding process is used because the negative mold 204 is
created from a material that can be dissolved or liquefied at a
temperature that does not affect a second fluid or gel contained
within the microchannels 207 of the negative mold 204. In the
preferred embodiment, the negative mold 204 is created from
polydimethylsiloxane (PDMS), but other dissolvable polymers can be
used.
[0036] In alternative embodiments, grooves 301 forming a vascular
network are machined directly onto the negative mold 204, obviating
the need for a master mold, or substrate 202. FIG. 17 is a series
of images of a lobule-mimetic vascular pattern directly machined on
smooth carboxymethlycellulose (CMC) sheets. The images in the first
column are scanning electron microscope characterizations. The
second column of images shows multiple negative molds 204 aligned
using precision guidance pins 302 and adhered to create a stacked,
three dimensional vascular network micromold 208.
[0037] After creating the negative molds 204, at step 102 a
micromold 208 is created by joining at least two negative molds
204. To ensure the cross-section of the space created by adjoining
grooves 301 has a circular cross-section, the two negative molds
204 must be precisely aligned. Alignment can be aided by the use of
a microscope and fiducial features. Alternatively, alignment pins
302 can be used. FIG. 16 shows a clamp 303 having alignment pins
302 disposed on one surface of the clamp 303 and guide rods 304. It
should be noted that for curved patterns, on each negative mold 204
the patterns will be mirror images of each other.
[0038] Next, at step 103, the vascular-mimetic micromolds 208 are
utilized to mold a vascular network template 205 by flowing a
fluid, or template material 209, into the microchannels 207 of the
micromold 208. The template material 209 is a material that can be
changed from a flowable to a hardened state, and then reversed to a
flowable state again. In the preferred embodiment, the material 209
is gelatin, a thermally reversible polymer. Gelatin is used because
it is (1) biocompatible or bioinert, (2) a solid phase at 4.degree.
C., (3) and a liquid at 37.degree. C. (body temperature). While
gelatin is used for the template material 209 in the preferred
embodiment, any thermally reversible polymer that is biocompatible,
in liquid form at body temperature (37.degree. C.), and becomes a
gel below room temperature (25.degree. C.) can be used. Examples of
thermally reversible polymers meeting these requirements include
polyester waxes and biodegradable copolymer compositions. A
thermally reversible polymer having a specific temperature profile
is used because--as will be explained in greater detail--using
higher temperatures could damage the cellular material contained in
a growth medium 206, in which the template 205 will be
embedded.
[0039] Once the template material 209 is flowed into the
microchannels 207 of the dissolvable micromold 208, the material
209 is hardened. In the preferred embodiment, the chemical
composition of the dissolvable polymer has a dissolution rate
chosen so that the dissolution of the micromold 208 does not occur
during fluid flow. In other words, the fluid can be flowed into the
microchannels 207 and cooled, forming a template 205 before the
micromold 208 dissolves and the microchannels 207 collapse. After
flowing the fluid into the microchannels 207, the temperature of
the micromold 208 and template material 209 is then decreased to
allow for complete gelation of the template material 209, and the
polymer micromold 208 is dissolved, thus leaving the structure of
the gelled or hardened template material 209, which serves as the
vascular template 205. FIG. 8A depicts the template material 209
flowed into the micromold 208 in the image on the left, and the
template 205 after dissolving the micromold 208 and hardening the
template material 209 in the image on the right.
[0040] In the preferred embodiment, in which a thermally reversible
polymer is used as the template material 209, the temperature of
the micromold 208 and fluid are reduced to about 4.degree. C.
However, the temperatures can vary depending on the materials used
for the micromolds 208 and template material 209.
[0041] At step 104, the biologic template 205 is then embedded
within a hepatocyte-seeded collagen gel 206, for example, to create
a vascular microarchitecture within the growth medium scaffold.
Depending on the tissue to be used with the vascular structure,
other types of cells can be seeded in the collagen gel or other
growth medium 206. This embedding step 104 is performed near room
temperature so that the vascular template 205, made of a thermally
reversible polymer, is maintained as a solid, while allowing for
collagen pre-gelation.
[0042] After embedding the template 205, at step 105, the
temperature is increased to allow for complete gelation of the
growth medium 206 and melting of the vascular template 205 back to
liquid form, creating a perfusable vascular cavity 210 within the
scaffold. In the preferred embodiment, the growth medium 206 is a
collagen material and the temperature is increased to 37.degree.
C., a temperature at which the thermally reversible polymer becomes
a liquid.
[0043] Finally, at step 106 cellular material is perfused into the
cavity 210. In the preferred embodiment, endothelial cells (ECs)
are seeded into the perfusable cavity 210 within the gelled
collagen scaffold 206 to form vascularized co-culture construct.
FIG. 8B shows the various stages of the construct. The image at the
left of FIG. 8B shows the template 205 embedded in a growth medium
206 containing hepatocytes. In the middle image, the template 206
has been liquefied and the cavity 210 perfused with a liquid
containing a cellular material, such as endothelial cells for
example. The image at the right of FIG. 8B shows endothelial cells
growing on the inner surface of cavity 210, wherein the endothelial
cells are surrounded by hepatocytes in the growth medium 206.
[0044] Endothelial cells are used as an example of cells to be used
in creating vascular tissue in the preferred embodiment. However,
the method can be used to create other web-like tissues, such as
nerves. If the method is being used to create innervated tissue
scaffolding, cells such as neurons, axons, and other neural cells
would be used instead of endothelial cells.
[0045] To assess the viability of a biologic construct, various
analytical techniques can be used. For example, FIG. 9 shows
various images of fluorescent beads flowed through cavity 210. The
bottom image in the first column shows a single layer vascular
network co-culture system that contains hepatocytes seeded within
the collagen gel 206 and endothelial cells cultured inside the
vascular channel comprising cavity 210, shown in two views: top x-y
plane and side x-z plane. A three-dimensional reconstruction of the
construct via fluorescent confocal microscopy shows
vascular-mimetic architecture and close interactions between
hepatocytes and endothelial cells. Moreover, endothelial cells and
hepatocytes remain viable within the collagen construct after
seeding.
[0046] In one embodiment of the method, hepatocytes and endothelial
cells can be seeded within the growth medium 206, or micropatterned
type I collagen hydrogels in this particular embodiment, during
cavity 210 fabrication. First, hepatocytes are suspended within the
collagen solution prior to gelation at an optimized seeding density
(5.times.106 cells per mL) in order to obtain uniform and adequate
distribution of hepatocytes (HepG2 cells) throughout the collagen
scaffold growth medium 206. Next, after one or two days of
culturing the hepatocyte-seeded vascular construct, endothelial
cells (ECs) (specifically HUVECs) are seeded into the vascular
channel, or cavity 210, in two-step process--first, a concentrated
solution of endothelial cells is injected into the cavity 210 and
incubated for a period of 12-24 hours to allow for cell attachment
on the bottom half of the cavity 210, and then a second
concentrated solution of endothelial cells is injected into the
cavity 210 and subsequently inverted to allow attachment to the top
half of the cavity 210.
[0047] To visually confirm attachment of the two cell types,
hepatocytes can be labeled with CellTracker Orange, which remains
within the cytoplasm of stained cells, and endothelial cells can be
labeled with wheat germ agglutinin-488 (WGA-488), which binds
specific lectin glycoproteins on the endothelial cell surface.
[0048] Vascular architecture and cellular distribution are assessed
via fluorescent confocal microscopy. Referring again to FIG. 9, the
single-layer vascular channel within the collagen construct is the
expected diameter, roughly 1 mm, as indicated by the presence of
fluorescent endothelial cells across the cavity 210 diameter
(marker by the dashed lines; top view of projected intensities on
the x-y plane). Confocal reconstructions reveal that hepatocytes
and endothelial cells are well distributed within the appropriate
locations. Hepatocytes have a uniform distribution through the
volume of the collagen gel and are in direct contact with the
endothelial cells lining the channel (side view of protected
intensities on the x-z plane). In addition, reconstructions of the
second image and bottom image in the second column in FIG. 9 of the
vascular cavity 210 reveal that endothelial cells are attached to
and cover the majority of cavity 210 interior surfaces one day
post-seeding. Confocal stacks are analyzed to give projected cell
intensity across the channel diameter as a means to assess cellular
distribution.
[0049] Cell viability of both hepatocytes and endothelial cells
within the collagen construct can be assessed through a Live/Dead
Cell Viability assay, shown in the bottom two images in the third
column of FIG. 9. After one day in culture after final HUVEC
seeding, HUVECS on both the top and bottom surfaces of the collagen
cavity 210 remain viable during the seeding and cell attachment
process. Hepatocytes within the collagen gel growth medium 206
closest to fresh nutrients and media remain viable over time;
however, hepatocytes in the bulk collagen gel do not remain fully
viable, likely due to lack of vascularization.
[0050] Cell tracking using live cell imaging dyes is another
analysis that can be performed to assess the construct. For
example, cellular morphology, migration, and proliferation over
time can be tracked through the use of live cell stains. The use of
Invitrogen CellTracker dyes can be used to label live cells prior
to seeding within the co-culture system (i.e. hepatocytes could be
pre-labeled orange, while endothelial cells could be pre-labeled
green). Several different CellTracker dye concentrations to ensure
viability of hepatocytes and endothelial cells in culture can be
used. As shown in FIG. 10, HepG2 cells are stained with three
concentrations of CellTracker Green BODIPY (Invitrogen)--0.5 .mu.M,
1 .mu.M, and 5 .mu.M--for 45 minutes, which resulting in a range of
fluorescence intensities. However, a dye concentration between 1-5
.mu.M is optimal, as the lowest concentration (0.5 .mu.M) results
in weak fluorescent signals over time. 5 .mu.M working solutions
for all CellTracker dyes (Orange CMTMR, Green BODIPY, and Blue
CMHC) for staining HepG2 cells are shown in FIG. 10; FIG. 11, and
HUVECs in FIG. 12.
[0051] Alternative methods of specific cell labeling can be used to
confirm that cells remain viable to allow for optimal functionality
of cells within a tissue-engineered system. These alternative
methods included WGA-488 (Wheat Germ Agglutinin, Alexa Fluor.RTM.
488 Conjugate; Invitrogen) labeling of HUVECs prior to cell
seeding. In addition, two stains can be used to image cells
immediately prior to imaging. CellMask Orange (Invitrogen) at 5
.mu.g/mL in PBS for 10 minutes can be used to label the plasma
membrane of HepG2 cells and/or HUVECS immediately before imaging,
as shown in the image in the middle column, top row of FIG. 12.
Hoescht 33432 (Invitrogen) at 2 .mu.g/mL in PBS for 2 minutes can
be used to label the nucleus of HepG2 cells and/or HUVECS prior to
imaging, as shown in the image in the last column, top row of FIG.
12.
[0052] To assess localization and abundance of cells seeded within
the vascularized co-culture construct, distribution of cells around
the channel can be assessed via quantitative image analysis.
Confocal stacks through the cell-populated channel are projected
onto the x-z plans, as depicted in FIG. 13. This allows for
analysis of project fluorescent intensity values as a metric of
hepatocyte and endothelial cell distributions across the cavity 210
diameter (x-axis). If the cells were seeded in the expected
locations, but in too low of a density (as shown in FIG. 13), cell
seeding can be improved by concentrating both cell types by
centrifugation and resuspension in a small volume of medium prior
to cell seeding. The concentration of hepatocytes within the
collagen solution can be increased a significant amount from
7.times.105 cells per mL to 2-5.times.106 cells per mL. To improve
endothelial cell seeding within the cavity 210, cells can be seeded
in a two-step process.
[0053] The method so far described can be used to fabricate
three-dimensional vascularized tissues using a PDMS as a mold 204
material. In an alternative embodiment, carboxymethlycellulose
(CMC), a water-soluble polymer is used as the polymer for the mold
204. This fabrication method can be scaled to build a
three-dimensional channel network within CMC, and the structures
micromachined in poly(methyl methacrylate) (PMMA), a polymeric
material, and used to mold a stackable three-dimensional CMC
channel system, as shown in FIG. 14. This three-dimensional CMC
microfluidic channel network can be used to mold three-dimensional
gelatin vascular templates 205. In this example, high strength (250
Bloom) gelatin at a concentration of 20% in distilled, deionized
water is injected in hot liquid form into a single layer CMC
channel and gelled at 4.degree. C. for at least 30 minutes.
[0054] To release the three-dimensional channel, the CMC structure
is placed in cold water, such that the CMC dissolves to leave
behind a solid three-dimension gelatin template 205. Next, this
gelatin template 205 is embedded within a hepatocyte-laden collagen
gel 206 (as demonstrated with the single layer channel), and upon
incubation near body temperature, the liquefied gelatin is flushed
out to allow for seeding of endothelial cells within the channel
network. In an alternative embodiment, direct fabrication of CMC
micromolds 208 can be achieved through machining the
vascular-mimetic patters directly onto a CMC substrate 202.
[0055] For complex three-dimensional shapes, an alternative
thermally reversible material having increased stiffness as
compared to gelatin, such as a low melting point polyester wax, can
be used. FIG. 18 shows a template 205 constructed of polyester wax,
which holds its shape after de-molding.
[0056] For proper operation of the stacked fluid network,
individual stacks must be sealed to create a fully circular
microchannel 207, so that assemblies between the layers can be
obtained. FIG. 15 shows an assembled channel, where, by sealing
with a liquid layer of CMC (FIG. 15, left image) as opposed to
water (FIG. 15, right image), channel 207 formation can be achieved
without cracks. This method allows for stacking of multiple layers
of CMC channels 207 that are well connected and perfusable. For
this purpose, successive steps of wetting are used (using a CMC
slurry between the channels) and load the assembly at high
temperatures (>120.degree. C.).
[0057] In addition to improved sealing between CMC layers, a
precision alignment platform to enable accurate assembly of
multiple CMC microchannels 207 through a layer-by-layer approach
can be used. One embodiment of the assembly apparatus is shown in
FIG. 16, with alignment pins 302 visible. Precision holes created
on the CMC sheets are aligned with the pins 302, enabling assembly
of each layer with single micron accuracy.
[0058] The methods and approach described herein can be extended to
build other tissue platforms including cardiac, circulatory,
endocrine, gastrointestinal, immune, integument, musculoskeletal,
nervous, reproductive, respiratory, and urinary. The major
components of this engineered platform can be applied to other
tissues enabling the: (1) control of vasculature within co-culture
systems; (2) ability to dictate structural position of multiple
different cell types within the tissue in spatially localized and
organized cell populations; (3) use of our conduits to introduce
additional cell types such as neural cells in defined positions;
(4) ability to scale-up significantly for larger scale production;
and (5) design of modules with specific diagnostic function
including integrated sensors and optical access for high throughput
and/or high content screening.
[0059] While the disclosure has been described in detail and with
reference to specific embodiments thereof, it will be apparent to
one skilled in the art that various changes and modifications can
be made therein without departing from the spirit and scope of the
embodiments. Thus, it is intended that the present disclosure cover
the modifications and variations of this disclosure provided they
come within the scope of the appended claims and their
equivalents.
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