U.S. patent application number 16/143050 was filed with the patent office on 2019-01-24 for method of printing a tissue construct with embedded vasculature.
This patent application is currently assigned to President and Fellows of Harvard College. The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Amelia Sydney GLADMAN, Kimberly A. HOMAN, David B. KOLESKY, Jennifer A. LEWIS, Mark A. SKYLAR-SCOTT, Ryan L. TRUBY.
Application Number | 20190022283 16/143050 |
Document ID | / |
Family ID | 53041985 |
Filed Date | 2019-01-24 |
View All Diagrams
United States Patent
Application |
20190022283 |
Kind Code |
A1 |
LEWIS; Jennifer A. ; et
al. |
January 24, 2019 |
METHOD OF PRINTING A TISSUE CONSTRUCT WITH EMBEDDED VASCULATURE
Abstract
A printed tissue construct comprises one or more tissue
patterns, where each tissue pattern comprises a plurality of viable
cells of one or more predetermined cell types. A network of
vascular channels interpenetrates the one or more tissue patterns.
An extracellular matrix composition at least partially surrounds
the one or more tissue patterns and the network of vascular
channels. A method of printing a tissue construct with embedded
vasculature comprises depositing one or more cell-laden filaments,
each comprising a plurality of viable cells, on a substrate to form
one or more tissue patterns. Each of the one or more tissue
patterns comprises one or more predetermined cell types. One or
more sacrificial filaments, each comprising a fugitive ink, are
deposited on the substrate to form a vascular pattern
interpenetrating the one or more tissue patterns. The vascular
pattern and the one or more tissue patterns are at least partially
surrounded with an extracellular matrix composition. The fugitive
ink is then removed to create vascular channels in the
extracellular matrix composition, thereby forming an
interpenetrating vascular network in a tissue construct.
Inventors: |
LEWIS; Jennifer A.;
(Cambridge, MA) ; KOLESKY; David B.; (Cambridge,
MA) ; SKYLAR-SCOTT; Mark A.; (Brookline, MA) ;
HOMAN; Kimberly A.; (Somerville, MA) ; TRUBY; Ryan
L.; (Boston, MA) ; GLADMAN; Amelia Sydney;
(Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
53041985 |
Appl. No.: |
16/143050 |
Filed: |
September 26, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15146613 |
May 4, 2016 |
10117968 |
|
|
16143050 |
|
|
|
|
PCT/US14/63810 |
Nov 4, 2014 |
|
|
|
15146613 |
|
|
|
|
61900029 |
Nov 5, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 33/00 20130101;
B29C 64/118 20170801; A61L 27/3808 20130101; B33Y 10/00 20141201;
A61L 27/3826 20130101; A61F 2/06 20130101; A61L 27/3813 20130101;
A61L 27/3633 20130101; A61F 2/105 20130101; A61F 2240/002 20130101;
B29C 64/40 20170801; A61L 2430/34 20130101; B33Y 80/00 20141201;
A61L 27/507 20130101; A61L 27/222 20130101; A61L 27/225
20130101 |
International
Class: |
A61L 27/50 20060101
A61L027/50; A61L 27/22 20060101 A61L027/22; B33Y 80/00 20150101
B33Y080/00; B29C 64/40 20170101 B29C064/40; A61L 27/38 20060101
A61L027/38; A61L 27/36 20060101 A61L027/36; B33Y 10/00 20150101
B33Y010/00; A61F 2/10 20060101 A61F002/10; A61F 2/06 20130101
A61F002/06; B29C 64/106 20170101 B29C064/106 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0003] This invention was made with government support under
contract number DMR 0820484 awarded by the National Science
Foundation. The government has certain rights in the invention.
Claims
1.-24. (canceled)
25. A three-dimensionally (3D) printed tissue construct with
embedded vasculature, the printed tissue construct comprising: one
or more tissue patterns, each tissue pattern comprising a plurality
of viable cells of one or more predetermined cell types produced by
depositing on a substrate one or more cell-laden filaments each
comprising a plurality of viable cells: a network of interconnected
vascular channels of various sizes interpenetrating the one or more
tissue patterns, wherein the lamest channels of the network provide
a single inlet and a single outlet for perfusion, while the
smallest channels of the network reduce the characteristic
diffusion distance between adjacent conduits; and an extracellular
matrix composition at least partially surrounding the one or more
tissue patterns and the interconnected network of vascular
channels.
26. The 3D printed tissue construct of claim 25, comprising up to n
predetermined cell types, where n is a positive integer satisfying
2.ltoreq.n.ltoreq.300.
27. The 3D printed tissue construct of claim 25, wherein the viable
cells are mammalian cells selected from the group consisting of:
germ cells, somatic cells and stem cells.
28. The 3D printed tissue construct of claim 25, wherein the
extracellular matrix composition comprises a synthetic or naturally
derived biomaterial.
29. The 3D printed tissue construct of claim 28, wherein the
extracellular matrix composition comprises a hydrogel.
30. The 3D printed tissue construct of claim 28, wherein the
extracellular matrix composition comprises at least one of gelatin,
fibrin, or gelatin methacrylate.
31. The 3D printed tissue construct of claim 30, wherein the
extracellular matrix composition further comprises a microgel.
32. The 3D printed tissue construct of claim 25, wherein the
extracellular matrix composition fully surrounds the network of
vascular channels.
33. The 3D printed tissue construct of claim 25, wherein the viable
cells are distributed uniformly throughout each of the one or more
tissue patterns.
34. The 3D printed tissue construct of claim 25, wherein at least
some of the viable cells of each of the one or more tissue patterns
are disposed at a distance of no greater than about 1 mm from one
or more of the vascular channels.
35. The 3D printed tissue construct of claim 34, wherein the
distance is no greater than about 300 microns.
36. The 3D printed tissue construct of claim 25, wherein each of
the one or more tissue patterns is defined by an arrangement of one
or more cell-laden filaments comprising the viable cells.
37. The 3D printed tissue construct of claim 36, wherein the one or
more cell-laden filaments further comprise an extracellular matrix
material.
38. The 3D printed tissue construct of claim 37, wherein the
extracellular matrix material comprises one or more of gelatin,
fibrin, and gelatin methacrylate.
39. The 3D printed tissue construct of claim 25, wherein at least
one of the one or more cell-laden filaments further comprise one or
more functional chemical substances selected from the group
consisting of: drugs, small molecules, toxins, proteins, and
hormones.
40. The 3D printed tissue construct of claim 39, wherein the
extracellular matrix composition comprises the one or more
additives, the one or more additives having diffused from the one
or more cell-laden filaments.
41. The 3D printed tissue construct of claim 25, wherein the
interconnected network of vascular channels has a branching
structure, the interconnected network comprising one or more
bifurcations.
42. The 3D printed tissue construct of claim 25, wherein one or
more of the vascular channels is curvilinear.
43. The 3D printed tissue construct of claim 25, wherein one or
more of the vascular channels has a nonuniform diameter along the
length thereof.
44. The 3D printed tissue construct of claim 25, wherein one or
more of the vascular channels comprise an endothelial layer
thereon, the endothelial layer comprising at least about 70%
confluency.
45. The 3D printed tissue construct of claim 44, wherein the
endothelial layer comprises 100% confluency.
46. The 3D printed tissue construct of claim 45, wherein one or
more of the vascular channels further comprise a stromal layer or a
smooth muscle cell layer on the endothelial layer.
47. The 3D printed tissue construct of claim 25, further comprising
one or more functional channels in the extracellular matrix
composition.
48. The 3D printed tissue construct of claim 47, wherein the one or
more functional channels comprise an epithelial layer thereon.
49. The 3D printed tissue construct of claim 48, wherein the one or
more functional channels further comprise a stromal layer on the
epithelial layer.
50. The 3D printed tissue construct of claim 25, further comprising
an interface structure at least partially surrounding the
extracellular matrix composition and comprising flow channels in
fluid communication with the interconnected network of vascular
channels.
51-56. (canceled)
57. The 3D printed tissue construct of claim 25, further comprising
one or more functional channels comprising an epithelial layer
thereon, wherein the extracellular matrix composition at least
partially surrounds the one or more functional channels.
58. The 3D printed tissue construct of claim 57, wherein the one or
more functional channels further comprises a stromal layer on the
epithelial layer.
59. (canceled)
60. The 3D printed tissue construct of claim 27, wherein the viable
cells and the one or more predetermined cell types comprise
epithelial cells.
61. (canceled)
62. The 3D printed tissue construct of claim 57, wherein the tissue
construct is an epithelial tissue construct selected from the group
consisting of nephron, intestine, milk duct, urethra, pancreatic
duct, and lymph.
Description
RELATED APPLICATIONS
[0001] The present patent document claims the benefit of priority
under 35 U.S.C. .sctn. 119 to U.S. Provisional Patent Application
Ser. No. 61/900,029, which was filed on Nov. 5, 2013, and which is
hereby incorporated by reference in its entirety.
[0002] The following patents and patent application publications
are also hereby incorporated by reference in their entirety:
International Patent Application Serial No. PCT/US2012/044794,
entitled "Multinozzle Deposition System for Direct Write
Applications," filed Jun. 29, 2012; U.S. Patent Application
Publication No. 2013/0084449, entitled "Viscoelastic Ink for Direct
Writing of Hydrogel Structures," which was filed as
PCT/US2011/29429 on Mar. 22, 2011; and U.S. Pat. No. 8,101,139,
entitled "Microcapillary Networks," filed on Jun. 5, 2008.
TECHNICAL FIELD
[0004] The present disclosure is related generally to tissue
engineering and more particularly to fabricating tissue constructs
including embedded vasculature.
BACKGROUND
[0005] The ability to create three-dimensional (3D) vascularized
tissues on demand could enable scientific and technological
advances in tissue engineering, drug screening, toxicology, 3D
tissue culture, and organ repair. To produce 3D engineered tissue
constructs that mimic natural tissues and, ultimately, organs,
several key components--cells, extracellular matrix (ECM), and
vasculature--may need to be assembled in complex arrangements. Each
of these components plays a vital role: cells are the basic unit of
all living systems, ECM provides structural support, and vascular
networks provide efficient nutrient and waste transport,
temperature regulation, delivery of factors, and long-range
signaling routes. Without perfusable vasculature within a few
hundred microns of each cell, three-dimensional tissues may quickly
develop necrotic regions. The inability to embed vascular networks
in tissue constructs has hindered progress on 3D tissue engineering
for decades.
BRIEF SUMMARY
[0006] A printed tissue construct comprises one or more tissue
patterns, where each tissue pattern comprises a plurality of viable
cells of one or more predetermined cell types. A network of
vascular channels interpenetrates the one or more tissue patterns.
An extracellular matrix composition at least partially surrounds
the one or more tissue patterns and the network of vascular
channels.
[0007] A method of printing a tissue construct with embedded
vasculature comprises depositing one or more cell-laden filaments,
each comprising a plurality of viable cells, on a substrate to form
one or more tissue patterns. Each of the one or more tissue
patterns comprises one or more predetermined cell types. One or
more sacrificial filaments, each comprising a fugitive ink, are
deposited on the substrate to form a vascular pattern
interpenetrating the one or more tissue patterns. The vascular
pattern and the one or more tissue patterns are at least partially
surrounded with an extracellular matrix composition. The fugitive
ink is then removed to create vascular channels in the
extracellular matrix composition, thereby forming an
interpenetrating vascular network in a tissue construct.
[0008] A method of printing an epithelial tissue construct entails
depositing one or more sacrificial filaments on a substrate to form
a functional channel pattern. Each of the sacrificial filaments
comprises a fugitive ink and a plurality of epithelial cells. The
functional channel pattern is at least partly surrounded with an
extracellular matrix composition. The fugitive ink is then removed
to create one or more functional channels in the extracellular
matrix composition. At least a portion of the epithelial cells
remain in the one or more functional channels after removal of the
fugitive ink, thereby forming an epithelial tissue construct.
[0009] A printed epithelial tissue construct comprises one or more
functional channels comprising an epithelial layer thereon. An
extracellular matrix composition at least partially surrounds the
one or more functional channels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1A shows an illustration of a bioprinting concept in
which vasculature, an extracellular matrix, and cells may be
printed in combination.
[0011] FIG. 1B shows a schematic of 3D printed heterogeneous tissue
construct that includes vasculature and multiple cell types
precisely placed in three dimensions.
[0012] FIG. 2A is a cross-sectional schematic showing a 2D tissue
construct including two tissue patterns and an interpenetrating
vascular network.
[0013] FIG. 2B shows an example of a 3D tissue construct including
a tissue pattern and an interpenetrating vascular network.
[0014] FIG. 2C shows an example of a 3D tissue construct including
two tissue patterns and an interpenetrating vascular network.
[0015] FIGS. 3A and 3B show examples of 3D tissue constructs
including two tissue patterns and an interpenetrating vascular
network.
[0016] FIG. 4 shows four print heads (nozzles) mounted onto a
custom 3D printer where each z-axis is controlled
independently.
[0017] FIG. 5A also shows four print heads (nozzles), and FIG. 5B
shows a four-layer microstructure where complex ink patterns are
deposited sequentially from four nozzles to form the four-layer
microstructure with varied composition.
[0018] FIG. 5C shows sequential fabrication images of each layer of
the deposition process, where each inset illustrates the geometry
of each layer.
[0019] FIG. 6A shows a schematic of the sol-gel transition of
Pluronic F127.
[0020] FIG. 6B shows the temperature dependence on the shear moduli
(G' and G'') of 40 wt. % Pluronic F127 ink.
[0021] FIG. 6C shows a schematic of the helix-to-coil transition
characteristic of GelMA.
[0022] FIG. 6D shows the thermal dependence of GelMA shear
moduli.
[0023] FIG. 6E shows GelMA laden with cells.
[0024] FIG. 6F shows the shear moduli as function of temperature
for GelMA laden with 10T1/2 fibroblast cells.
[0025] FIGS. 7A and 7B show 1D channels formed in GelMA, where
diameters range from 115 .mu.m to 500 .mu.m.
[0026] FIG. 7C shows the channels perfused with a water-soluble
fluorescent dye for visualization.
[0027] FIGS. 7D-7E show a 2D hierarchical branching network with
curvilinear filaments printed using a single 30 .mu.m glass
capillary.
[0028] FIG. 7F shows the structure perfused with red fluorescent
dye for visualization.
[0029] FIGS. 7G-7I show a highly periodic 3D lattice printed from
sacrificial filaments to create a 3D vascular pattern that can be
perfused after evacuation of the fugitive ink.
[0030] FIG. 7J shows an optical image of representative
microchannel within a 2D vascular network perfused with a HUVEC
suspension.
[0031] FIG. 7K shows a confocal image of the microchannel shown in
FIG. 7J with live HUVEC cells lining the microchannel walls.
[0032] FIG. 8A shows representative cross-sections of various
channel diameters created by depositing sacrificial filaments and
removing the fugitive ink.
[0033] FIG. 8B is a plot showing the swelling ratio
(D.sub.final/D.sub.initial) of various printed sacrificial
filaments comprising Pluronic F127. Each data point is an average
of six samples, each deposited at a fixed speed, pressure, and
nozzle height (z axis). The diameters are measured directly after
printing and again after evacuation via top-down optical
microscopy.
[0034] FIGS. 9A-9B show that endothelialized vascular channels can
be created in fibrin gel, as shown.
[0035] FIGS. 9C and 9D show before and after photographs of animal
blood infiltration in a fabricated bifurcating vascular
network.
[0036] FIG. 10A provides a top-down view of a final 3D printed
heterogeneous tissue constructs structure that is printed from four
separate inks.
[0037] FIG. 10B provides an angled view of the complex tool-path
used to create the tissue construct shown in FIG. 10A, where the
green filaments comprise GFP HNDF-laden GelMA, the blue filaments
comprise 10T1/2 fibroblast-laden GelMA, and the red filaments
comprise the Pluronic ink that may be endothelialized with RFP
HUVECs. The gray shaded region corresponds to pure GelMA matrix
that encapsulates the 3D printed construct.
[0038] FIG. 10C is a bright field microscopy image overlaid with
the green fluorescent channel of the structure of FIG. 10A directly
after printing.
[0039] FIG. 10D is a photograph illustrating the spanning and
out-of-plane nature of the printed structure.
[0040] FIG. 10E shows a demonstration of the fugitive ink
evacuation process.
[0041] FIG. 10F provides a composite image of the three fluorescent
channels: 10T1/2 fibroblasts (blue), HNDFs (green), HUVECs (red)
from the structure of FIG. 10A.
[0042] FIG. 10G shows cell-viability assay results of printed
10T1/2 fibroblasts compared with a non-printed control.
[0043] FIG. 11A shows a schematic of a three-layered structure
containing multiple cell-laden filaments and sacrificial filaments
comprising a fugitive ink.
[0044] FIG. 11B shows a photograph of the evacuated microstructure
before endothelialization.
[0045] FIG. 11C shows an epifluorescent image of GFP HNDFs (green)
and RFP HUVECs (red) after two days in culture.
[0046] FIGS. 12A-12C show a schematic and images that illustrate
the shaping of growth factor (GF) gradients by direct printing of a
GF-laden extracellular matrix material comprising
fibrin-gelatin.
[0047] FIG. 12D show an exemplary mold for perfusion of parallel
printed vascular channels with fluorescently labeled BSA in only
one channel.
[0048] FIGS. 12E and 12F show that a nearly linear gradient is
generated between the two channels of FIG. 12D in 24 hours.
[0049] FIGS. 13A-13D illustrate the synthesis of a fibrin-gelatin
interpenetrating polymer network. First, gel precursors are mixed
together with transglutaminase (TG). Then, by polymerizing
fibrinogen via the enzyme thrombin, a fibrin network is formed. The
second phase is then formed around the fibrin gel, and the two
phases are slowly crosslinked together via TG.
[0050] FIGS. 13E-13F show mechanical properties of the
fibrin-gelatin matrix material.
[0051] FIGS. 13G-13J show the diversity of the fibrin-gelatin
matrix adhesivity for fibroblasts (connective tissue), smooth
muscle cells, endothelial cells, and renal proximal tubule
(epithelial) cells, respectively.
[0052] FIG. 14 illustrates the influence of TG incubation time on
the optical properties of the fibrin-gelatin interpenetrating
polymer network. Transparency is determined by the final pore
architecture of the fibrin gel, which is visualized using a
rhodamine tagged fibrinogen and confocal microscopy.
[0053] FIGS. 15A-15D show an exemplary mold for passive rocking
perfusion of a tissue construct.
[0054] FIGS. 15E-15G show exemplary molds for active pump-based
perfusion of a tissue construct.
[0055] FIGS. 16A-16C show schematically the deposition of
endothelial cells within a sacrificial filament formed from a
fugitive ink, encapsulation of the sacrificial filament with an
extracellular matrix composition, and evacuation of the fugitive
ink to form a channel with endothelial cells lining the channel
wall.
[0056] FIG. 16D shows an as-printed fugitive ink (Pluronic F127)
comprising a dispersion of HUVECs; FIG. 16E shows the fugitive ink
after casting and liquefying; FIG. 16F shows the vascular network
after 1 day of incubation of the HUVECs; and FIG. 16G shows the
vascular network after active perfusion for 24 hours.
[0057] FIG. 17A illustrates the creation of one or more functional
channels in an extracellular matrix composition to form a
functional channel network in a tissue construct (specifically, in
this example, an epithelial tissue construct). The steps include
deposition of one or more sacrificial filaments comprising a
fugitive ink (which may be a cell-laden fugitive ink) to form a
functional channel pattern, at least partial encapsulation of the
channel pattern with an extracellular matrix composition, removal
of the fugitive ink to form the functional channels, and an
optional seeding approach for lining the functional channels with
epithelial cells.
[0058] FIGS. 17B-17C shows two functional channels in an
extracellular matrix composition where the channels are lined with
epithelial cells.
[0059] FIGS. 17D-17F show various confocal microscopy images of
PTEC-lined channels and immunofluorescence images with various
cell-specific proteins being expressed, including Na/K ATPase.
[0060] FIGS. 18A-18C highlight the initially uniform distribution
of HUVECs (red) and HNDFs (green) at 3 days post-seeding. FIGS.
18D-18E shows the same channel after eight days, at which time the
channel comprises a distinct outer stromal (HNDF) layer and a
confluent endothelial (HUVEC) layer.
[0061] FIGS. 19A-19F show that, after printing a fugitive ink
directly onto a cell-laden matrix, encapsulating with more
cell-laden matrix, evacuating the fugitive ink to form vascular
channels, and seeding the vascular channels with HUVECs, the
endothelial cells form confluent layers and assemble into capillary
structures over time.
[0062] FIGS. 20A-20C are confocal microscopy images that show
spontaneous neovasculature formation in a printed cell-laden
filament comprising two cell types (HNDFs and HUVECs dispersed
within a gelatin-fibrin extracellular matrix material).
[0063] FIG. 21A shows a schematic depicting an embedded printing
process; FIG. 21B shows a schematic of an extracellular matrix
composition comprising a semi-interpenetrating polymer network
(IPN) (e.g., PAA-GelMA) suitable for embedded printing; and FIG.
21C illustrates a complex heterogeneous structures with arbitrary
3D shape that may be constructed by embedded printing.
[0064] FIG. 21D provides representative rheological measurements of
ink and matrix rheology appropriate for embedded printing; FIG. 21E
is a photograph of a vascular cube including a vascular network
formed by embedded printing; and FIG. 21F shows a printed
cell-laden filament within a semi-IPN extracellular matrix
composition.
[0065] FIG. 22 demonstrates that the transparency of a semi-IPN
extracellular matrix composition may be tuned by degree of
substitution (DS).
DETAILED DESCRIPTION
[0066] A printed tissue construct including an interpenetrating
vasculature and a method of printing such a tissue construct are
described herein. FIG. 1A provides an illustration of the
bioprinting concept. The printing method may enable the fabrication
of heterogeneous 2D and 3D tissue constructs including cells,
vasculature, epithelial ducts, and extracellular matrix in
predetermined locations for applications ranging from 3D tissue
culture and drug screen to organ transplants.
[0067] FIG. 1B and FIGS. 2A-2C provide schematics showing exemplary
printed tissue constructs that include vasculature and multiple
cell types precisely placed in three dimensions. Referring to FIG.
2A or 2C, an exemplary printed tissue construct 100 comprises a
first tissue pattern 115a and a second tissue pattern 115b, where
each of the first and second tissue patterns 115a,115b comprises a
plurality of viable cells of one or more predetermined cell types.
For example, the first tissue pattern 115a may include cell types A
and B, and the second tissue pattern 115b may include cell type C.
An arrangement of one or more cell-laden filaments 105 comprising
the viable cells and having the predetermined cell types may define
each tissue pattern 115a,115b. In this example, the cell-laden
filaments 105 that define the first tissue pattern 115a include
cell types A and cell B, and the cell-laden filaments that define
the second tissue pattern 115b include cell type C. A network of
vascular channels 135 interpenetrates the tissue patterns
115a,115b. An extracellular matrix composition 130 at least
partially surrounds the one or more tissue patterns 115a,115b and
the network of vascular channels 135.
[0068] A pattern or network that "interpenetrates" another pattern
or network in a printed tissue construct may be understood to
comprise one or more filaments, channels or portions that are
layered with, partially or completely overlapping, partially or
completely underlapping, surrounding, embedded within, and/or
interwoven with one or more filaments, channels or portions of the
other pattern or network. A filament "deposited on a substrate" may
be understood to be deposited directly on the substrate or directly
on another filament, channel or portion previously deposited or
formed on the substrate.
[0069] Referring now to FIG. 2B, a tissue construct comprising an
embedded vasculature may be printed by depositing one or more
cell-laden filaments 105, where each cell-laden filament 105
comprises a plurality of viable cells, on a substrate 110 to form
one or more tissue patterns 115 (one tissue pattern in this
example). The tissue pattern 115 comprises cells of one or more
predetermined cell types. One or more sacrificial filaments 120,
each comprising a fugitive ink, are also deposited on the substrate
110 to form a vascular pattern 125 that interpenetrates the one or
more tissue patterns 115. The one or more tissue patterns 115 and
the vascular pattern 125 are partially or fully surrounded by an
extracellular matrix composition 130. The fugitive ink is then
removed to create a network of vascular channels 135 in the
extracellular matrix composition 130. Thus, an interpenetrating
vascular network is formed in the tissue construct 100.
[0070] The tissue construct may include up to n different
predetermined cell types. For example, n may satisfy
1.ltoreq.n.ltoreq.300, 2.ltoreq.n.ltoreq.200, or
2.ltoreq.n.ltoreq.100. More typically, n is no more than 50, no
more than 30, or no more than 20. For example, there may be 2 or
more, 4 or more, 8 or more, 16 or more, or 20 or more predetermined
cell types in the tissue construct.
[0071] As illustrated by the examples of FIGS. 2A-2C, each tissue
pattern comprises or is defined by a two- or three-dimensional
arrangement of one or more cell-laden filaments, and each tissue
pattern (and thus each arrangement of cell-laden filaments) may
comprise a different subset of the predetermined cell types. For
example, in a tissue construct that includes 5 different
predetermined cell types (e.g., cell types A, B, C, D, and E) and 3
different tissue patterns (e.g., tissue patterns 1, 2, and 3),
tissue pattern 1, which is defined by a first arrangement of one or
more cell-laden filaments, may comprise cell type A; tissue pattern
2, which is defined by a second arrangement of one or more
cell-laden filaments, may comprise cell types B and C; and tissue
pattern 3, which is defined by a third arrangement of one or more
cell-laden filaments, may comprise cell types A and E.
[0072] In addition to the viable cells, the one or more cell-laden
filaments may comprise a synthetic or naturally-derived
biocompatible material that may be referred to as an extracellular
matrix material. Each of the one or more cell-laden filaments may
also or alternatively comprise one or more functional chemical
substances (e.g., drugs, toxins, proteins and/or hormones) as
described below. Each tissue pattern may include one layer or
multiple layers of the cell-laden filament(s), which may in some
embodiments be at least partially coalesced at regions of contact
therebetween. For example, adjacent layers formed from one or more
cell-laden filaments may be partially or fully coalesced depending
on filament composition and the deposition (or post-deposition)
conditions.
[0073] The arrangement of the cell-laden filaments in the tissue
construct may be continuous or discontinuous. In a continuous
arrangement, the cell-laden filaments of an exemplary tissue
pattern (and comprising one or more predetermined cell types) may
form a single interconnected tissue network in the tissue
construct. For example, a single cell-laden filament comprising
viable cells of the predetermined cell type(s) may be deposited in
a single layer or in multiple layers to form the continuous
arrangement. Alternatively, a plurality of cell-laden filaments
comprising viable cells of the predetermined cell type(s) may be
deposited in a single layer or in multiple layers to form the
continuous arrangement, where each of the cell-laden filaments is
in physical contact with, and possibly at least partially coalesced
with, another cell-laden filament comprising the same predetermined
cell type(s).
[0074] In a discontinuous arrangement of cell-laden filaments
comprising one or more predetermined cell types, a single
interconnected tissue network of the predetermined cell type(s) is
not formed within the tissue construct. Instead, the cell-laden
filaments comprising the predetermined cell type(s) may be
dispersed uniformly or nonuniformly throughout the tissue
construct. Consequently, the cells corresponding to the
predetermined cell type(s) may also be dispersed uniformly or
nonuniformly (e.g., in clumps) throughout the tissue construct. In
this embodiment, some, all or none of the cell-laden filaments of a
given tissue pattern and cell type(s) may be in physical contact
with another cell-laden filament comprising cells of the same cell
type(s).
[0075] Each of the one or more cell-laden filaments includes at
least one viable cell and may include a large number of viable
cells. For example, each of the cell-laden filaments may have a
cell concentration of at least about 100 cells/ml, at least about
1000 cells/ml, at least about 10.sup.4 cells/ml, at least about
10.sup.5 cells/ml, at least about 10.sup.6 cells/ml, at least about
10.sup.7 cells/ml, or at least about 10.sup.6 cells/ml. Typically,
the cell concentration is no higher than about 10.sup.9 cells/ml,
or no higher than about 10.sup.8 cells/ml. Consistent with this,
the one or more tissue patterns of the tissue construct may have a
cell concentration of at least about 100 cells/ml, at least about
1000 cells/ml, at least about 10.sup.4 cells/ml, at least about
10.sup.5 cells/ml, at least about 10.sup.6 cells/ml, at least about
10.sup.7 cells/ml, or at least about 10.sup.8 cells/ml. Typically,
the cell concentration in the tissue pattern is no higher than
about 10.sup.8 cells/ml, or no higher than about 10.sup.8
cells/ml.
[0076] The cell concentration may be substantially uniform (e.g.,
within .+-.10%, within .+-.5%, or within .+-.1%) throughout each of
the cell-laden filaments, and the cell concentration may also be
substantially uniform throughout each of the tissue pattern(s).
Alternatively, it is possible to deposit cell-laden filaments that
include aggregates or dusters of cells that may range in size from
about 10 cells/duster to about 1000 cells/cluster, or from about 10
cells/cluster to about 100 cells/duster. Such dusters may be
dispersed uniformly or non-uniformly within the cell-laden
filaments (and thus uniformly or non-uniformly throughout the one
or more tissue patterns). Overall, the cell concentration may be
substantially uniform throughout the tissue construct, or the cell
concentration may include predetermined inhomogeneities within the
tissue construct that may be defined by the location and morphology
of the one or more tissue patterns, and/or by the cell distribution
within the one or more tissue patterns.
[0077] The vascular network that interpenetrates the one or more
tissue patterns is a two- or three-dimensional interconnected
arrangement of vascular channels. The network may include one or
more -furcations (e.g., bifurcations, trifurcations, etc.) from a
parent vascular channel to a plurality of branching vascular
channels. The network may have a hierarchical branching structure,
where larger diameter channels branch into smaller diameter
channels. Some or all of the vascular channels may follow a curved
path, and thus may be considered to be curvilinear. All of the
vascular channels in the network may have the same diameter, or at
least one, some, or all of the vascular channels may have a
different diameter. In some cases, one or more of the vascular
channels may have a nonuniform diameter along a length thereof.
[0078] It is beneficial for the cells of the tissue construct to be
close enough to the interpenetrating network of vascular channels
to remain viable. One major problem with previous attempts to
create tissue and organ-like structures is that necrotic regions
could develop in areas without accessible perfusable vasculature.
In the present work, each cell-laden filament, and thus each cell,
may be placed in a location near to the vascular network, or near
to where the vascular network may be formed. For example, at least
a portion of the one or more cell-laden filaments forming each
tissue patter, and thus some or all of the viable cells, may be no
more than about 1 mm away, no more than about 500 microns away, no
more than about 300 microns away, no more than about 200 microns
away, no more than about 100 microns away, no more than about 50
microns away, and/or no more than about 10 microns away from a
vascular channel. One or more of the cell-laden filaments and thus
at least some of the viable cells may be deposited so as to be in
direct contact with a vascular channel. It is envisioned that some
portion of the vascular network, for example the smallest
capillaries, may be formed by angiogenesis and/or tubulogenesis
after deposition of the sacrificial filaments and removal of the
fugitive ink. For example, cell-laden filaments comprising
endothelial cells may be deposited adjacent to the fugitive network
to encourage tubulogenesis and/or angiogenesis to generate new
capillaries.
[0079] Because the printing process described below for deposition
of the cell-laden (and other) filaments allows for a high
positional accuracy, the placement of the viable cells and/or the
extracellular matrix material within the tissue construct may be
controlled to within .+-.200 microns, within .+-.100 microns,
within .+-.50 microns, within .+-.10 microns, or within .+-.1
micron.
[0080] Different types of cells may be placed in close proximity to
one another by depositing a cell-laden filament that includes cells
of more than one cell type, as discussed above. It is also
contemplated that, in addition to the interpenetrating vasculature,
one or more of the tissue patterns may interpenetrate one or more
of the other tissue patterns so that certain types of cells may be
positioned in close proximity to another. For example, one or more
cell-laden filaments comprising a first type of cells (e.g.,
epithelial or endothelial cells) may be layered with, partially or
completely overlapping, partially or completely underlapping,
surrounding, embedded within, and/or interwoven with one or more
cell-laden filaments comprising a second type of cells (e.g.,
smooth muscle cells). In some embodiments, all of the tissue
patterns may interpenetrate at least one other tissue pattern, and
it is also contemplated that all of the tissue patterns may
interpenetrate all of the other tissue patterns.
[0081] The extracellular matrix composition may partially or fully
surround the one or more tissue patterns, where a tissue pattern
that is fully surrounded includes no exposed cell-laden filaments.
The extracellular matrix composition may also partially or fully
surround the network of vascular channels, where a vascular network
that is fully surrounded includes no exposed vascular channels. For
example, the network of vascular channels may be fully surrounded
by the extracellular matrix composition, while the tissue pattern
may be only partially surrounded by (e.g., adjacent to) the
extracellular matrix composition. In such an example, the
cell-laden filaments may be deposited after the vascular pattern is
encapsulated. In some embodiments, the extracellular matrix
composition may comprise additional viable cells and/or one or more
functional chemical substances, as described below, which may be
deposited along with the extracellular matrix composition. Such an
extracellular matrix composition may be referred to as a cell-laden
matrix. As described below, the extracellular matrix composition
may be printed, cast or formed by another method known to one of
ordinary skill in the art.
[0082] The tissue construct may have any desired 2D or 3D shape.
For example, the tissue construct may have a planar geometry
constructed from a single layer or multiple layers of cell-laden
filaments and an interpenetrating vascular network. Such structures
may have any desired height (thickness). Typically, the tissue
construct has a height of about 100 cm or less, about 10 cm or
less, about 1 cm or less, about 1 mm or less, about 500 microns or
less, or about 100 microns or less, and typically at least about 10
microns, at least about 100 microns, at least about 200 microns, or
at least about 1 mm, with applications ranging from tissue cultures
and drug screening to skin constructs and corneal replacements.
[0083] Alternatively, the tissue construct into which a vascular
network is embedded may have an arbitrary or application-dependent
3D size and shape. The tissue construct may have a solid structure,
a porous structure, and/or a hollow structure (e.g., tubular or
nontubular) and may be fabricated to mimic the morphology and
function of particular organ. For example, the tissue construct may
have the size and shape of a kidney, heart, pancreas, liver,
bladder, vagina, urethra, trachea, esophagus, skin or other bodily
organ. The 3D size and shape may in some cases be determined by a
mold, as described below.
[0084] In general, in a three-dimensional arrangement of cell-laden
filaments with an interpenetrating vascular pattern, the
sacrificial filaments may have portions that overlie or underlie
portions of the cell-laden filaments, and the sacrificial and
cell-laden filaments may or may not be confined to an XY plane
normal to the vertical direction (as defined by the force of
gravity). The sacrificial filaments may be in physical contact with
some or all of the cell-laden filaments, and, in some embodiments,
the filaments may be partially or fully coalesced at the regions of
contact. Both the sacrificial and cell-laden filaments may have
spanning portions that extend unsupported between points of
contact.
[0085] FIGS. 2A-2C show exemplary tissue constructs 100 each
comprising one or more tissue patterns 115 interpenetrated by a
vascular pattern 125 or a network of vascular channels 135. In FIG.
2A, two tissue patterns 115a,115b each comprising two cell-laden
filaments 105 are deposited on the substrate 110 in a single layer.
Adjacent to and/or in physical contact with the cell-laden
filaments 105 are sacrificial filaments 120 of the vascular pattern
125, where each sacrificial filament 120 comprises a fugitive ink.
After encapsulation with the extracellular matrix composition 130,
the fugitive ink may be removed to form the network of vascular
channels 135.
[0086] FIG. 2B shows a schematic of a tissue construct 100
comprising a 3D lattice structure 115 of cell-laden filaments 105
alternating with sacrificial filaments 120 of an interpenetrating
vascular pattern 125. The fugitive ink making up the sacrificial
filaments 120 is ultimately removed to create the network of
vascular channels 135, which may also be visualized in FIG. 2B.
[0087] The tissue construct 100 of FIG. 2C includes two tissue
patterns 115a,115b each defined by a curvilinear cell-laden
filament 105 that are interpenetrated by a network of vascular
channels 135 (or by a vascular pattern 125 comprising sacrificial
filaments 120 if the fugitive ink has not yet been removed). The
vascular network 135 has a hierarchical branching structure
including curvilinear channels of various lengths and diameters. A
solid substrate 110 is shown underlying the tissue construct 100;
however, in this and in the other exemplary figures, the underlying
solid substrate 110 may not be present.
[0088] FIG. 3A shows a top view of two exemplary tissue patterns
115a,115b comprising cell-laden filaments 105 in a semi-woven
configuration with sacrificial filaments 120 of a vascular pattern
125. FIG. 3B shows multiple layers of the same tissue patterns
115a,115b and vascular pattern 125 (or network of vascular channels
135 if the fugitive ink has been removed) surrounded by an
extracellular matrix composition 130.
[0089] The viable cells and the predetermined cell types in the
tissue construct may include any mammalian cell type selected from
cells that make up the mammalian body, including germ cells,
somatic cells, and stem cells. Depending on the type of cell, cells
that make up the mammalian body can be derived from one of the
three primary germ cell layers in the very early embryo: endoderm,
ectoderm or mesoderm. The term "germ cells" refers to any line of
cells that give rise to gametes (eggs and sperm). The term "somatic
cells" refers to any biological cells forming the body of a
multicellular organism; any cell other than a gamete, germ cell,
gametocyte or undifferentiated stem cell. For example, in mammals,
somatic cells make up all the internal organs, skin, bones, blood
and connective tissue. As such, a cell may include any somatic cell
isolated from mammalian tissue, including organs, skin, bones,
blood and connective tissue (i.e., stromal cells). Examples of
somatic cells include fibroblasts, chondrocytes, osteoblasts,
tendon cells, mast cells, wandering cells, immune cells, pericytes,
inflammatory cells, endothelial cells, myocytes (cardiac, skeletal
and smooth muscle cells), adipocytes (i.e., lipocytes or fat
cells), parenchyma cells (neurons and glial cells, nephron cells,
hepatocytes, pancreatic cells, lung parenchyma cells) and
non-parenchymal cells (e.g., sinusoidal hepatic endothelial cells,
Kupffer cells and hepatic stellate cells). The term "stem cells"
refers to cells that have the ability to divide for indefinite
periods and to give rise to virtually all of the tissues of the
mammalian body, including specialized cells. The stem cells include
pluripotent cells, which upon undergoing further specialization
become multipotent progenitor cells that can give rise to
functional or somatic cells. Examples of stem and progenitor cells
include hematopoietic stem cells (adult stem cells; i.e.,
hemocytoblasts) from the bone marrow that give rise to red blood
cells, white blood cells, and platelets; mesenchymal stem cells
(adult stem cells) from the bone marrow that give rise to stromal
cells, fat cells, and types of bone cells; epithelial stem cells
(progenitor cells) that give rise to the various types of skin
cells; neural stem cells and neural progenitor cells that give rise
to neuronal and glial cells; and muscle satellite cells (progenitor
cells) that contribute to differentiated muscle tissue.
[0090] The tissue construct may also include one or more functional
chemical substances selected from among drugs, toxins, proteins
and/or hormones, including, but not limited to: growth factors,
growth inhibitors, cytokines, steroids, and/or morphogens. Some
cell specific examples include: bone morphogenic protein, vascular
endothelial growth factor, fibroblast growth factors, including but
not limited to VEGF, EGF, TGF-beta. The one or more functional
chemical substances may be deposited with the cell-laden
filament(s) and/or the sacrificial filaments and may diffuse into
the surrounding extracellular matrix composition.
[0091] Such an approach may be used to generate gradients of cues
within the extracellular matrix composition. Cells respond to
gradients of fixed and diffusible chemical cues during development,
wound healing and inflammatory responses that can direct cell
migration, proliferation and differentiation. One method to
introduce gradients of cues is to directly print cell-laden
filaments preloaded with cues of interest, as illustrated in FIGS.
12A-12C, which may diffuse out upon encapsulation with an
extracellular matrix composition to generate concentration
gradients. Such gradients may or may not be anchored to the
scaffold by action of transglutaminase. Alternatively, to generate
fixed, long-term gradients, the channels formed by removing
fugitive ink can be used to introduce factors that may diffuse into
the surrounding extracellular matrix composition. For example, the
formation of a linear gradient of fluorescently labeled BSA is
demonstrated in FIGS. 12D-12F by creating a pair of parallel
channels and flowing the fluorescent BSA through only one channel.
At 24 hours, a near-linear gradient is apparent between the two
channels (FIGS. 12E-12F).
[0092] The extracellular matrix material of the cell-laden
filaments and the extracellular matrix composition that at least
partially surrounds the tissue and vascular patterns may comprise a
synthetic or naturally derived biocompatible material. The
extracellular matrix material and the extracellular matrix
composition may comprise the same or different biocompatible
materials. Because the cell-laden filaments and, in some
embodiments, the extracellular matrix composition may be deposited
in a 3D printing process that entails extrusion through a
micronozzle, as described below, it may be beneficial for one or
both of the extracellular matrix material and the extracellular
matrix composition to: (1) exhibit shear thinning behavior; (2)
exhibit a defined yield stress .tau..sub.y; and/or (3) have a shear
elastic modulus G' and a shear viscous modulus G'' modulus where
G'>G'' at room temperature.
[0093] In one example, the extracellular matrix material and/or the
extracellular matrix composition may comprise a gel. An ideal gel
for bioprinting applications may exhibit a rapid transition from a
low viscosity solution to a solid-like gel, which may be seen by an
initial increase in shear elastic modulus. Rapid, controllable
gelation may enhance printed structure fidelity by minimizing or
obviating swelling and dissociation typical of slow gelation
processes. The term "gel" may refer to a semi-solid substance that
may comprise a gelling agent to provide viscosity or stiffness. The
gel may be formed upon use of a gelling agent, such as a thickening
agent, crosslinking agent or a polymerization agent, and may
comprise a cross-linked structure or a non-cross-linked structure.
The gel may be hydrophobic or hydrophilic. Some examples of
suitable gels include a hydrogel, thermo-reversible gel, a
photo-sensitive gel, a pH sensitive gel, a peptide gel, or a cell
type specific gel. Additional examples of gels include silica gel,
silicone gel, aloe vera gel, agarose gel, nafion, polyurethane,
elastomers (thermoplastic, mineral-oil thermoplastic, etc.),
ion-exchange beads, organogels, xerogels and hydrocolloids.
Hydrogels include those derived from collagen, hyaluronate, fibrin,
alginate, agarose, chitosan, gelatin, matrigel, glycosaminoglycans,
and combinations thereof. In one example, the gel may comprise
gelatin methacrylate (GelMA), which is denatured collagen that is
modified with photopolymerizable methacrylate (MA) groups. Suitable
hydrogels may comprise a synthetic polymer. In certain embodiments,
hydrogels may include those derived from poly(acrylic acid) and
derivatives thereof, poly(ethylene oxide) and copolymers thereof,
poly(vinyl alcohol), polyphosphazene, and combinations thereof. The
extracellular matrix material and/or the extracellular matrix
composition may comprise a naturally derived biocompatible
material, such as one or more extracellular matrix components,
including collagen, fibronectin, laminin, hyaluronates, elastin,
and/or proteoglycans. Other suitable biocompatible materials for
the extracellular matrix material and/or the extracellular matrix
composition may include variations of cellulose, Matrigel,
acrylates, acrylamides, polylactic co-glycolic acid, epoxies,
aldehydes, ureas, alcohols, polyesters, silk, proteins,
glycosaminoglycans, carbohydrates, minerals, salts, clays,
hydroxyapatite, and/or calcium phosphate.
[0094] In a preferred embodiment, the extracellular matrix material
and/or the extracellular matrix composition may comprise gelatin
and fibrin. The gelatin and fibrin may form an interpenetrating
polymer network that mimics natural extracellular matrix (ECM) and
may be optimized for cell attachment, bioprinting, transparency,
and biocompatibility. The fibrin-gelatin interpenetrating polymer
network may be created by mixing solutions of fibrinogen and
gelatin with transglutaminase (TG), a slow-acting Ca.sup.2+
dependent enzyme, to create a gel-precursor solution that may later
be mixed with bovine thrombin to create a fibrin gel backbone, as
illustrated in FIGS. 13A-13D. Fibrin may be made from a
concentrated fibrinogen solution that has been activated by bovine
thrombin and calcium chloride. Fibrin is a rapidly coagulating
phase that permits rapid, controllable gelation of a printed
structure. Advantageously, fibrin and gelatin can be welded
together via mobile surface chain entanglement, while forming a
strong interface. Creating monolithic gels of this nature is
possible due to the slow crosslinking kinetics of transglutaminase
(TG). Although thrombin rapidly induces fibrin gel formation, the
gelatin present in the IPN allows one to print sacrificial ink on
the already cast layer, and, ultimately, to encapsulate with liquid
gelatin-fibrin. The two phases may weld together, creating a
monolithic gel. This material system, which is discussed further
below in the Examples, can be readily tailored to modify gelation
kinetics, interface adhesion, mechanical properties, optical
properties, and cell-material interactions.
[0095] As described above, one or more sacrificial filaments
comprising a fugitive ink may be deposited on a substrate to form a
vascular pattern that interpenetrates one or more tissue patterns.
The vascular pattern comprises a two- or three-dimensional
interconnected arrangement or network of the one or more
sacrificial filaments. Removal of the fugitive ink after partial or
complete encapsulation with the extracellular matrix composition
creates a perfusable network of vascular channels in the tissue
construct. Because, like the cell-laden filaments, the sacrificial
filaments may be deposited in a 3D printing process that involves
extrusion through a micronozzle, it may be advantageous for the
fugitive ink to: (1) exhibit shear thinning behavior; (2) exhibit a
defined yield stress .tau..sub.y; and/or (3) have a shear elastic
modulus G' and a shear viscous modulus G'' modulus where G'>G''
at room temperature.
[0096] The substrate for deposition typically comprises a material
such as glass or other ceramics, PDMS, acrylic, polyurethane,
polystyrene or other polymers. In some embodiments, the substrate
may comprise living tissue or dehydrated tissue, or one of the
extracellular matrix compositions described above. The substrate
may be cleaned and surface treated prior to printing. For example,
glass substrates may undergo a silane treatment to promote bonding
of the cell-laden filaments to the glass substrate. In some
embodiments, it is envisioned that the substrate may not be a
solid-phase material but may instead be in the liquid or gel phase
and may have carefully controlled rheological properties, as
described, for example, in W. Wu et al., Adv. Mater. 23 (2011)
H178-H183, which is hereby incorporated by reference. In the work
of Wu et al., a fugitive ink was printed directly into synthetic
hydrogels to create network structures. However, these synthetic
materials do not support cell attachment and proliferation,
limiting their use to non-biological applications. In the present
disclosure, an extracellular matrix composition that facilitates
cell attachment, migration, proliferation, and tissue-specific
function while maintaining the appropriate rheology for printing is
described. The cell-laden and sacrificial filaments are embedded in
the extracellular matrix composition during printing, and thus the
at least partial surrounding of the tissue and vascular patterns
with the extracellular matrix composition occurs during deposition
of each of the cell-laden and sacrificial filaments, as shown
schematically in FIG. 21A. This includes arbitrarily complex 3D
structures that may require support material during printing, as
shown for example in FIG. 21C. When the forming and embedding of
the tissue and vascular patterns occurs simultaneously, as
described above, the substrate onto which deposition occurs may be
considered to be the container that holds the extracellular matrix
composition or the extracellular matrix composition itself.
[0097] To form the extracellular matrix composition, a microgel
(e.g., a poly(acrylic acid) (PAA) microgel) may be used as a
rheological modifier and blended with one or more extracellular
matrix materials, as set forth previously, such as gelatin
methacrylate. A semi-interpenetrating polymer network (semi-IPN)
may be formed, as shown schematically in FIG. 21B. Microgels may be
understood to comprise colloidal gel particles that are composed of
chemically cross-linked three-dimensional polymer networks.
Microgels may act as sterically stabilized colloids with only a
shell and no core. They can vary in composition and may include
PAA, polystyrenes, PEG, and/or other biomaterials. It is
contemplated that a natural extracellular matrix or biomaterial may
be converted into a microgel form to impart the ideal rheology.
Examples of suitable biomaterials include hyaluron, collagen,
alginate, fibrin, albumin, fibronectin, elastin, or matrigel.
Alternatively, synthetic materials such as PEG, acrylates,
urethanes, or silicones may be modified in a similar manner.
[0098] Representative rheological measurements of ink and matrix
rheology that are appropriate for embedded printing are shown in
FIG. 21D. In one example, a high molecular weight (>1.25 MDa)
PAA microgel is used as a rheological modifier and blended with
gelatin-methacrylate (GelMa) to create an extracellular matrix
composition that supports the creation of complex 3D vascular
networks, which can be endothelialized as described previously. The
transparency of the extracellular matrix composition may be altered
by varying the degree of substitution and mesh size, as shown in
FIG. 22. FIG. 21E shows a vascular cube demonstrating the control
over the embedded printing of 3D vascular networks, and FIG. 21F
shows a printed cell-laden filament within a PAA-GelMA
extracellular matrix composition.
[0099] The method may further include, prior to surrounding or
encapsulating the tissue and vascular patterns with the
extracellular matrix composition, depositing one or more structural
filaments layer by layer on the substrate in a predetermined
pattern to form a mold. The structural filaments may comprise one
or more structural materials selected from among the exemplary
extracellular matrix compositions or extracellular matrix materials
provided above. The mold may hold the extracellular matrix
composition during the encapsulation and may remain as part of the
tissue construct, or it may be removed after processing. The
structural filaments may define the perimeter of the tissue
construct on the substrate and all or at least a portion of the
three-dimensional shape of the tissue construct out of the XY
plane.
[0100] The mold may also have other functionalities besides
defining the shape of the tissue construct. For example, the mold
may serve as an interface for perfusion of channels in a printed
tissue construct. FIGS. 15A-15D and 15E-15G show exemplary designs
of printed molds or interface structures. The exemplary mold shown
in FIGS. 15A-15D is designed for passive rocking perfusion. The
mold, which may also be referred to as an interface structure, can
hold vascularized tissue in place during rocking by immobilizing
the tissue construct between a base portion of the mold, which may
comprise PDMS, and an overlying cover, which may comprise
glass.
[0101] The mold designs of FIGS. 15E-15G enable active pump-based
perfusion of a tissue construct and include flow channels that are
in fluid communication with (e.g., contiguous with) the vascular
channels of the tissue construct. Conduits that serve as flow
channels may be partially or fully embedded in the mold itself and
hollow pins (e.g., metal pins) may be used to interface with the
vascular channels, as shown in FIGS. 15F-15G. The exemplary mold
shown in FIG. 15E has a wall with multiple buttresses that contain
the flow channels, which include hollow pins extending into the
interior of the mold, where the tissue construct is fabricated. The
vascular channels of the tissue construct may be contiguous with
apertures of the hollow pins to enable flow to be introduced into
the vascular channels from tubing connected to the flow channels,
and fluid may be removed from the vascular channels through one or
more other apertures.
[0102] In one example, the mold may be formed of an elastomeric
silicone, a structural material known to be viscoelastic,
non-toxic, biocompatible, and capable of forming reversible
press-to-fit seals. The structural material may be 3D printed to
form one or more uncured structural filaments comprising one or
more of silicone, epoxies, esters of acrylic acid, or one of the
extracellular matrix compositions provided above. After printing is
complete, the structural filament(s) may be cured (e.g. by heating
or photopolymerizing) for a suitable time duration (e.g., about one
hour or more), after which the mold may exhibit the desired
material properties.
[0103] The encapsulation of the tissue and vascular patterns may
comprise casting a liquified matrix precursor into the mold and
gelling the matrix precursor to form the extracellular matrix
composition. Casting of the matrix precursor may take place at a
temperature of from about 25.degree. C. to about 40.degree. C. For
example, gelatin methacrylate, or GelMA, may be cast at a
temperature of about 37.degree. C. After casting, the matrix
precursor may be cooled (e.g., to about 15.degree. C. in the case
of GelMA) to form a rigid physical gel. Alternatively, the
encapsulation may occur during deposition of the tissue and
vascular patterns in an embedded or omni-directional 3D printing
process, as indicated above. It is also contemplated that the
extracellular matrix composition may be deposited by filament
deposition, similar to the cell-laden and sacrificial filaments.
For example, one or more ECM filaments comprising the extracellular
matrix composition may be extruded from a nozzle and deposited on
the substrate layer by layer to build up the desired 3D geometry,
as described below. In such a case, it may not be necessary to
employ a mold to contain the extracellular matrix composition.
[0104] The extracellular matrix composition may be cured before or
after removal of the fugitive ink to form a permanently chemically
cross-linked structure. Depending on the extracellular matrix
composition, the curing may entail heating, UV radiation or
chemical additives (e.g., enzymatic curing).
[0105] Any or all of the filaments deposited on the
substrate--including the cell-laden filaments defining the one or
more tissue patterns, the one or more sacrificial filaments
defining the interpenetrating vascular pattern or a functional
channel pattern, the one or more structural filaments that may
define the mold, and/or the one or more ECM filaments that may
yield the extracellular matrix composition--may be extruded from a
nozzle before being deposited on the substrate. In the discussion
of the extrusion process that follows, the sacrificial filaments,
the cell-laden filaments, the structural filaments and/or the ECM
filaments may be collectively referred to as "the filaments" since
the processing steps may be applicable to any or all of the
filament compositions.
[0106] FIG. 4 shows four exemplary nozzles or print heads that may
be employed to extrude the filaments and deposit them on the
substrate. The nozzles shown are part of a custom-built 3D printer
comprising a large build platform (750 mm.times.650 mm) equipped
with four independent z-axes. FIGS. 5A-5C provide a demonstration
of the 3D printing of a four-layer, multimaterial construct by
sequential deposition of a filament of a different composition from
each of the four nozzles. The insets of FIG. 5C show, for each
layer, the repeating unit of the 3D structure.
[0107] Although there are four nozzles for the exemplary printer of
FIGS. 4 and 5A, the number of nozzles employed to form the tissue
construct by 3D printing may be lower or higher. In general, 1 or
more, 2 or more, 3 or more, 4 or more, 5 or more and up to N
nozzles may be used for extruding the filaments, where
1.ltoreq.N.ltoreq.1024, and more typically N is no more than 512, N
is no more than 256, N is no more than 128, or N is no more than
64. The filaments may be extruded from the N nozzles sequentially
in a serial deposition process or simultaneously in a parallel
deposition process, where each nozzle may contain a different
precursor ink (e.g., a cell-laden ink comprising one or more
predetermined cell types, a fugitive ink, a structural ink, or an
ECM ink). It is also contemplated that the deposition may include
both parallel and serial deposition steps. To facilitate sequential
or serial printing, the nozzles can be independently controlled in
the z-direction, as shown in FIG. 4.
[0108] Each nozzle may have an inner diameter of from about 1
micron to about 1 mm in size, and more typically from about 50
microns to about 500 microns. The size of the nozzle may be
selected depending on the desired filament diameter. Depending on
the injection pressure and the nozzle translation speed, the
deposited filament may have a diameter ranging from about 1 micron
to about 10 mm, and more typically from about 100 microns (0.1 mm)
to about 1 mm. The inks fed to the nozzles may be housed in
separate syringe barrels that may be individually connected to a
nozzle for printing by way of a Luer-Lok.TM. or other connector.
The extrusion of each of the filaments may take place under an
applied pressure of from about 1 psi to about 200 psi, from about
10 psi to about 80 psi, or from about 20 psi to about 60 psi. The
pressure during extrusion may be constant or it may be varied. By
using alternative pressure sources, pressures of higher than 100
psi or 200 psi and/or less than 1 psi may be applied during
printing. A variable pressure may yield a filament having a
diameter that varies along the length of the filament. Such an
approach may be used, for example, to form the branching,
hierarchical vascular network shown in FIG. 2C and in FIGS. 7E-7F,
which is formed from sacrificial filaments of various lengths and
diameters. The extrusion may be carried out at ambient or room
temperature conditions (e.g., from about 18.degree. C. to about
25.degree. C.).
[0109] During the extrusion and deposition of each filament, the
nozzle may be moved along a predetermined path (e.g., from
(x.sub.1, y.sub.1, z.sub.1) to (x.sub.2, y.sub.2, z.sub.2)) with a
positional accuracy of within .+-.100 microns, within .+-.50
microns, within 110 microns, or within .+-.1 micron. Accordingly,
the filaments may be deposited with a positional accuracy of within
.+-.200 microns, within .+-.100 microns, within .+-.50 microns,
within .+-.10 microns, or within .+-.1 micron. The nozzles may be
moved and the filaments may be deposited at speeds as high as about
3 m/s (e.g., from about 1 cm/s to about 3 m/s), and are more
typically in the range of from about 1 mm/s to about 500 mm/s, from
about 1 mm/s to about 100 mm/s, or from about 1 mm/s to about 10
mm/s.
[0110] The predetermined path of the nozzle may have an XY boundary
area of at least about 2400 cm.sup.2, at least about 2700 cm.sup.2
and up to about 1 m.sup.2 as determined by the size of the build
platform of the printer. For example, the build platform may have a
length of from about 60 cm to about 100 cm and a width of from
about 40 cm to about 100 cm. Each print head may be moved in the
z-direction a distance from about 10 cm to about 50 cm, or about 15
to about 30 cm.
[0111] The deposited filaments are formed from precursor inks
(e.g., cell-laden inks comprising one or more predetermined cell
types, fugitive inks, structural inks, or ECM inks) having a
suitable composition and rheological properties. The precursor inks
may be viscoelastic and comprise a viscosity with a non-linear
shear dependence. The viscosity of the precursor inks may fall in
the range of from about 0.001 Pa-sec to about 10,000 Pa-sec. The
precursor inks may optionally include viscosifiers to help control
the rheological properties. Each cell-laden ink, and optionally,
the fugitive and/or ECM ink, may include one or more cells of one
or more predetermined cell types in a carrier that may be a liquid
or a gel. The carrier may include, in addition to an extracellular
matrix material as described above, one or more functional chemical
substances as described above. The carrier may also or
alternatively include a cell culture medium designed to support the
growth of cells. In one example, to form a cell-laden ink
comprising viable cells mixed with a hydrogel, a predetermined
amount of a hydrogel precursor powder is mixed with a cell culture
medium to form a solution of an appropriate composition. The cells
of interest are then dispersed in the solution at the desired cell
concentration (e.g., any of the cell concentrations set forth above
for the cell-laden filaments), and mixed thoroughly. Steps to
prepare exemplary cell-laden GelMA inks, cell-laden gelatin-fibrin
inks, Pluronic F127 fugitive inks, and PDMS structural inks are
described in the Examples below.
[0112] After encapsulation of the tissue and vascular patterns, the
fugitive ink may be removed to form a network of vascular channels
in the extracellular matrix composition. The fugitive ink may
comprise a biocompatible material and may be designed for
compatibility with the cell-laden formulations and the
extracellular matrix composition during room temperature
deposition. Suitable fugitive inks may include, for example,
Pluronic F127, Pluronic F123, agarose, sugar, wax, and fatty oils
(e.g., animal fat derived oils such as Crisco). If a hydrogel is
employed for the extracellular matrix composition (and/or the
extracellular matrix material), and a hydrogel such as Pluronic
F127 is employed as the fugitive ink, it may be advantageous for
the fugitive ink and the matrix hydrogel to have similar water
contents (e.g., within 130%) to avoid distortion of the fugitive
ink after printing. The fugitive ink and the extracellular matrix
composition may also be selected to have complementary thermal
transitions, as discussed further below.
[0113] Pluronic F127 is an FDA-approved material that is
biologically inert to multiple cell types over the short time
periods needed to complete the fabrication process. The material
includes a hydrophobic poly(propylene oxide) (PPO) segment and two
hydrophilic poly(ethylene oxide) (PEO) segments arranged in a
PEO-PPO-PEO configuration. Pluronic F127 undergoes thermally
reversible gelation above a critical micelle concentration (CMC;
about 21 wt. %) and the gelation temperature. The gelation
temperature decreases from approximately 10.degree. C. to 4.degree.
C. as the PEO-PPO-PEO concentration increases. When both of these
critical parameters are exceeded, micelles form as the hydrophilic
PEO segments self-assemble into corona that are well solvated by
water, while the hydrophobic PPO segments tightly associate within
the micelle cores. However, below the gelation temperature, the
hydrophobic PPO units are hydrated, such that individual
PEO-PPO-PEO species become soluble in water giving rise to a
gel-to-fluid transition for systems whose concentration exceeds the
CMC. Thus, the material liquefies upon cooling below the gel
point.
[0114] It is important that the patterned cells and surrounding
extracellular matrix composition are not damaged during deposition
of the sacrificial filaments or removal of the fugitive ink, and
thus it is preferred that harsh solvents and/or elevated
temperatures are not utilized during the removal process. With
proper selection of the fugitive ink and the extracellular matrix
composition/material, the fugitive ink may be removed without
damage to the tissue construct. For example, if the fugitive ink
undergoes a gel-to-fluid transition as described above, cooling of
the vascular pattern after encapsulation may be effective for
removal of the fugitive ink. To remove Pluronic F127, the vascular
pattern may be cooled to a temperature of no more than about
1.degree. C., depending on the concentration. It is also
contemplated that the fugitive ink may be dissolved in a suitable
aqueous solution for removal. Once the fugitive ink is liquefied or
dissolved, a vacuum may be applied to an exposed end of the
vascular pattern to extract the ink.
[0115] Advantageously, the tissue constructs may be designed to
support the attachment and proliferation of endothelial cells,
which line vascular channels providing a barrier to fluid
diffusion, while simultaneously facilitating homeostatic functions
and helping establish vascular niches specific to the various
tissues. To promote endothelialization, in some embodiments the
sacrificial filament(s) comprising the fugitive ink may further
include a plurality of endothelial cells or other viable cells. The
cells may be deposited along with the sacrificial filament and may
remain in the vascular channels after removal of the fugitive ink,
as illustrated in FIGS. 16A-16C. Direct cellularization of the
channels can be achieved if the cells adsorb to the channel walls
after liquidation of the fugitive ink. This approach may allow one
to incorporate viable cells into highly tortuous networks or small
channels that may be difficult to infill using direct injection due
to an increased resistance to flow. An exemplary printed tissue
construct including a channel formed by evacuation of a fugitive
ink comprising endothelial cells and Pluronic F127 is shown in
FIGS. 16D-16G, and is further described in the Examples. In another
example, epithelial cells may be delivered in a fugitive ink and
used to create tubular epithelial tissues present in the mammary
gland, kidney or liver.
[0116] In addition to or as an alternative to depositing
endothelial and/or other viable cells with the fugitive ink,
endothelialization may be effected by injecting a suspension of
viable cells (e.g., endothelial cells) into the vascular channels
after removing the fugitive ink. Using one or both of these
approaches, an endothelial layer having up to 100% confluency may
be formed lining the wall of one or more of the vascular channels,
where 100% confluency means that the wall is completely covered by
endothelial cells. Each endothelial layer formed in the network of
vascular channels may have a confluency of at least about 80%, at
least about 90%, at least about 95%, at least about 98%, at least
about 99%, or 100%, so that the vascular channels may function as
actual blood vessels. As described in the Examples below, it has
been shown that representative hierarchical bifurcating networks
may be successfully injected with a human umbilical vein
endothelial cell (HUVEC) suspension followed by gentle rocking (see
FIG. 7J). After 48 h, these cells retained greater than 95%
viability and assembled into a nearly confluent layer, as
determined by live/dead staining coupled with confocal imaging
within a representative, bifurcated microchannel.
[0117] Multiple types of cells may be injected into the vascular
channels. In vivo, every blood vessel having a diameter larger than
a capillary contains an outer fibrous tissue layer, a smooth muscle
layer, and an inner layer of endothelial cells. One or more other
types of cells, such as fibroblasts, may be injected into the
vascular channels along with the endothelial cells after removing
the fugitive ink. As described in the Examples below, the vascular
channels may be co-seeded with fibroblasts and HUVECs, two cell
types which may self-assemble into stromal and endothelial layers,
respectively, mimicking the anatomy of native blood vessels.
[0118] It is also contemplated that the same or a different
fugitive ink may be deposited as a sacrificial filament and removed
as described above to form channels, ducts and/or compartments in
addition to or in lieu of the vascular channels within the tissue
construct. In other words, one or more additional sacrificial
filaments may be deposited to form a functional channel pattern on
the substrate, either in addition to or in lieu of the vascular
pattern. This is shown schematically in FIG. 17A.
[0119] Each additional sacrificial filament may comprise a second
fugitive ink, which is the same as or different from the fugitive
ink used to define the vascular pattern (if the vascular pattern is
present). After deposition, the functional channel pattern may be
at least partially surrounded with the extracellular matrix
composition (e.g., the hydrogel solution shown in step III of FIG.
17A), as described above in reference to the vascular pattern. It
is also contemplated that the at least partial surrounding of the
functional channel pattern with the extracellular matrix
composition may occur during deposition of the one or more
sacrificial filaments, such that the one or more functional channel
patterns are simultaneously formed and embedded in the
extracellular matrix composition. When the forming and embedding of
the functional channel patterns occurs simultaneously, the
substrate onto which deposition occurs may be considered to be the
container that holds the extracellular matrix composition or the
extracellular matrix composition itself. The second fugitive ink
may then be removed, as illustrated in step V of FIG. 17A and as
described above in reference to the vascular channels, to create
one or more functional channels in the extracellular matrix
composition. Thus, a functional channel network may be formed in
the tissue construct, which in this example is an epithelial tissue
construct, as shown in FIGS. 17A (center) and 17B. One or more
types of viable cells (e.g., epithelial cells) may be deposited
with the additional sacrificial filaments, and at least a portion
of the viable cells may remain in the one or more functional
channels after removal of the second fugitive ink. Also or
alternatively, after removing the second fugitive ink, a suspension
of viable cells (e.g., epithelial cells) may be injected into the
functional channels, as shown in step VI of FIG. 17A.
[0120] The functional channels may define tubular tissues or tissue
components. Examples of tubular structures that can be formed via
3D printing and epithelialization include, but are not limited to,
a nephron (of the kidney), intestine, milk duct, urethra, and
lymph. Such a printed epithelial tissue construct may comprise one
or more functional channels comprising an epithelial layer thereon,
and an extracellular matrix composition may at least partially
surround the one or more functional channels, as illustrated in
FIGS. 17A (center) and 17B. A stromal layer may also be present on
the epithelial layer. The printed epithelial tissue construct may
further comprise one or more tissue patterns, each comprising a
plurality of viable cells of one or more predetermined cell types,
in the extracellular matrix composition, as set forth above. The
viable cells and the one or more predetermined cell types may
comprise epithelial cells and/or another cell type described
previously. The printed epithelial tissue construct may further
comprise a network of vascular channels in the extracellular matrix
composition, also as described above.
[0121] For example, a network of vessels (channels) of the
lymphatic system may be created using sacrificial filaments
comprising a fugitive ink. In another example, compartments of any
desired geometry may be embedded within the tissue construct by
depositing a predetermined arrangement of sacrificial filaments.
Such embedded compartments may be used for containing growth
factors, additional cells and/or supplemental scaffold materials
that may in some embodiments be deposited with the sacrificial
filaments to direct cell behavior, differentiation, function,
movement and/or growth.
[0122] A printed epithelial tissue construct comprising a
functional channel that is subsequently seeded with epithelial
cells (epithelialization) is shown in FIGS. 17A-17F. Referring to
FIGS. 17A-17B, the printed tissue construct is a proximal
convoluted tubule, a portion of the nephron. It can be printed in a
simple or convoluted shape and seeded with epithelial cells, which
thrive and circumscribe the functional channels, as shown in FIGS.
17C-17F. The cells employed are human renal proximal tubule cells
(PTEC); however, this approach may be applied to any of a number of
types of epithelial tissue. For example, in vitro models may be
fabricated for tissue-specific disease and toxicity studies. This
type of functional human tissue mimic can be used as a building
block for the growth of larger organs or for high throughput drug
toxicity and screening.
EXAMPLES
Fugitive Ink
[0123] Referring to FIG. 6A, highly concentrated (40 wt. %)
Pluronic F127A, which exhibits a strong shear-thinning response
when the applied shear stress exceeds the shear yield stress
(.tau..sub.y) (e.g., during printing), as well as a plateau shear
elastic modulus (G') that exceeds the shear viscous modulus (G'')
when the applied shear stress is below .tau..sub.y (e.g., after
printing), is selected as the fugitive ink for an exemplary system.
The fugitive ink elasticity is found to be about 2.times.10.sup.4
Pa at 22.degree. C., as shown in FIG. 6B. Below the CMT (about
4.degree. C.), the ink liquefies and its elasticity decreases by
several orders of magnitude, thereby facilitating its removal from
the tissue construct.
[0124] As described above, the sacrificial filaments formed from
the fugitive ink may include one or more additional cells, growth
factors, drugs, etc. For example, endothelial, epithelial and/or
other cells may be dispersed within the fugitive ink and deposited
with the sacrificial filaments. When the fugitive ink is removed to
form the vascular (or other) channels, the cells may remain, lining
walls of the channels.
[0125] This approach is demonstrated with a highly concentrated
endothelial cell-laden fugitive (pluronic) ink (1.times.10.sup.7
cells/ml). The fugitive ink is deposited and encapsulated with an
extracellular matrix composition. Upon removal of the fugitive ink
to form vascular channels, the endothelial cells remain affixed to
walls of the channels, as shown schematically in FIGS. 16A-16C and
experimentally in FIGS. 16D-16G. FIG. 16G shows a simple channel
created using this approach that has been perfused for over 24
hours. The endothelial cells appear to line the channel and look
qualitatively similar to those created using a conventional seeding
approach. This technique provides an alternative to seeding
existing vascular channels with endothelial cells, particularly in
the case of highly branched vascular networks where cells may clog
and inhibit flow, leading to non-uniform seeding.
Extracellular Matrix Composition and Material
[0126] As set forth above, an interpenetrating polymer network
based on gelatin and fibrin has been developed that mimics natural
ECM, and which may be used for the extracellular matrix composition
and/or the extracellular matrix material of the tissue
construct.
[0127] FIGS. 13A-13D show fabrication of a gelatin-fibrin
interpenetrating polymer network, or gelatin-fibrin matrix. First,
the gel precursors are first mixed together. Polymerizing
fibrinogen via the enzyme thrombin forms a fibrin gel or network.
This phase provides initial mechanical strength and rigidity, as
indicated by an increase in shear elastic modulus. The second phase
(gelatin) is then formed around the fibrin gel, and the two phases
are slowly crosslinked together via transglutaminase (TG). FIGS.
13E and 13F show shear modulus versus time (G' and G'') and a
stress-strain curve for the gelatin-fibrin interpenetrating polymer
network.
[0128] TG is a naturally occurring enzymatic protein crosslinker
with myriad biological functions; for example, it may be
up-regulated during wound healing in vivo. By varying TG incubation
time, the optical properties (e.g., transparency) of the fibrin gel
can be tailored. The transparency is dictated by the final pore
architecture of the fibrin gel, which is visualized using a
rhodamine-tagged fibrinogen and confocal microscopy. It is also of
interest to determine if TG and gelatin disrupt natural fibrin
polymerization. Confocal microscopy images reveal that the
fibrillar nature of fibrin is preserved and can be precisely tuned
by varying different processing conditions, such as incubation
time, as illustrated in FIG. 14. A longer fibrin-TG incubation time
leads to a more dense fibrillar network and, subsequently, higher
optical transparency.
[0129] Besides fabrication considerations, cell
material-interactions play an important role in materials
selection. The gelatin-fibrin matrix has been shown to be
compatible with many different cell types, including fibroblasts
(connective tissue), smooth muscle cells, endothelial cells, and
renal proximal tubule cells (epithelial). The adhesivity of the
gelatin-fibrin matrix has been quantified by comparing the
projected area of cells on various substrates. The gelatin-fibrin
matrix outperformed all other materials including native fibrin,
tissue culture polystyrene (TCPS), and gelatin methacrylate
(GelMa). FIGS. 13G-131 show various cells essential for creating
blood vessels--fibroblasts, endothelial cells, and smooth muscle
cells--on an exemplary gelatin-fibrin matrix surface. To highlight
the diversity of the adhesivity, tissue-specific epithelial cells
were grown on the surface, as illustrated in FIG. 13J.
[0130] In a second example, gelatin methacrylate (GelMA), which is
biocompatible, easily processed and inexpensive, is selected for
use as both the extracellular matrix material for the cell-laden
formulation and as the extracellular matrix composition for the
encapsulation step. GelMA is denatured collagen that is modified
with photopolymerizable methacrylate (MA) groups, which allows the
matrix to be covalently cross-linked by UV light after printing.
Physical gelation arises from the assembly of intermolecular triple
helices that possess a structure similar to collagen, as
illustrated in FIG. 6C. By varying the concentration, degree of
methacrylation, and temperature, the shear yield stress and elastic
modulus of aqueous GelMA systems can be systematically tuned.
[0131] The extracellular matrix composition is produced by
dissolving 15 wt. % GelMA in cell culture media. Above
approximately 25.degree. C., the composition is a low viscosity
fluid with a G' value below 101 Pa. Upon cooling below 25.degree.
C., the composition undergoes gelation, yielding a clear,
viscoelastic extracellular matrix material. The elasticity of the
extracellular matrix composition increases with decreasing
temperature, with G' values of about 10.sup.3 Pa and
2.times.10.sup.4 Pa observed at 22.degree. C. and 2.degree. C.
(FIG. 6D), which correspond to typical conditions for printing and
fugitive ink removal, respectively.
[0132] The same aqueous GelMA composition is used to create
cell-laden inks that contain viable cells for printing. Prior
studies have shown that cells adhere, remodel, and migrate through
GelMA due to the presence of integrin-binding motifs and matrix
metal-proteinase sensitive groups. It is found that the
incorporation of a moderate concentration, e.g., 2.times.10.sup.6
cells/mL, of 10T1/2 fibroblast cells into the 15 wt. % GelMA ink
(FIGS. 6E and 6F) does not significantly alter the temperature at
which gelation ensues or the elasticity of the composition over the
temperature range of interest, e.g., 2.degree. C. to 40.degree. C.
Hence, both pure and cell-laden GelMA inks can be printed and
further processed, as needed, in the same manner.
[0133] The differences in thermally reversible gelation observed
for the fugitive Pluronic F127, pure GelMA, and cell-laden GelMA
inks give rise to three distinct processing windows. Between
approximately 4.degree. C. and 25.degree. C., each ink is stiff and
exhibits a solid-like response, where G'>G''. At
T.gtoreq.25.degree. C., the Pluronic F127 fugitive ink is stiff and
solid-like (G'>G''), while the pure and cell-laden GelMA inks
are liquids that flow readily. Below about 4.degree. C., the
Pluronic F127 fugitive ink is a liquid that flows readily, while
the pure and cell-laden GelMA inks are stiff and solid-like
(G'>G'').
Printing of Vascular Patterns
[0134] The complimentary thermal behavior described above for the
Pluronic F127-GelMA system is exploited to print representative
vascular patterns comprising a plurality of sacrificial filaments
which are then encapsulated in an acellular extracellular matrix
composition (pure GelMA). FIGS. 7A-7K illustrate the formation of
1D, 2D and 3D vascular networks and endothelialization of the
channels, with schematic views and corresponding optical images of
each vascular network design. After removing the fugitive ink, each
vascular network is perfused with a fluorescent red dye to aid in
visualization (FIGS. 7C, 7F and 7I). Within each tissue construct,
the diameter of the sacrificial filaments can be altered as desired
by modifying the printing pressure, speed, and/or nozzle height.
For example, 1-D microchannel arrays with diameters increasing from
45 .mu.m to 500 .mu.m are printed using a single 30 .mu.m nozzle
simply by increasing the printing pressure and nozzle height in a
stepwise fashion between each printed feature (FIGS. 7A-7B).
[0135] After photopolymerizing the GelMA matrix, the fugitive ink
is removed by cooling the printed constructs below 4.degree. C.,
yielding open 1-D microchannels. Representative cross-sectional
images of these 100 .mu.m channels, shown in FIG. 8A, reveal that
their final diameters range from about 100 .mu.m to about 1 mm.
Since the GelMA ink has a higher water content than the fugitive
ink, the printed vascular features may swell as water diffuses into
the fugitive ink (Pluronic F127) from the surrounding matrix.
Indeed, the diameters nearly double in size, with a swelling ratio
that is independent of initial microchannel diameter (FIG. 8B) for
this material system.
[0136] The 2-D vascular network design mimics the hierarchical,
bifurcating motifs found in biological systems, large channels
bifurcate to form smaller channels that maximize efficient blood
flow, nutrient transport, and waste removal while minimizing the
metabolic cost. These 2D hierarchical vascular networks are printed
using a single nozzle of 30 microns (e.g., FIGS. 7E and 7F). The
as-printed, largest channels (650 .mu.m in diameter) provide a
single inlet and outlet for perfusion, while the smallest channels
(45 .mu.m) in diameter) reduce the characteristic diffusion
distance between adjacent conduits. Finally, a 3D microvascular
network design, which is shown in FIGS. 7G, 7H and 7I and includes
a 3D periodic array of uniform microchannels, is printed. Because
the embedded microchannels are interconnected in all three
dimensions, the fugitive ink can be removed from the surrounding
GelMA matrix quickly and with high fidelity.
Seeding of Vascular Channels
[0137] Multiple types of fluids may be flowed through embedded
vascular networks to demonstrate their perfusable nature. For
example, the 2D hierarchical bifurcating networks are injected with
a human umbilical vein endothelial cell (HUVEC) suspension followed
by gentle rocking. After 48 h, it is found that the cells retained
greater than 95% viability and assembled into a nearly confluent
layer, as determined by live/dead staining coupled with confocal
imaging within a representative, bifurcated microchannel.
[0138] The vascular channels may be seeded with multiple cell
types, such as fibroblasts or smooth muscle cells in addition to
HUVECs. Here, fibroblasts are co-seeded with HUVECs. It is found
that, after about one week of perfusion culture, uniformly
co-seeded endothelial cells and fibroblasts self assemble into two
distinct layers of outer enveloping stroma (human dermal
fibroblasts; HNDFs) and confluent inner endothelium (HUVECs). FIGS.
18A-18C highlight the initially uniform distribution of HUVECs
(red) and HNDFs (green) at 3 days post-seeding. FIGS. 18D-18E shows
the same channel after eight days, at which time the channel
comprises a distinct outer stromal (HNDF) layer and a confluent
endothelial (HUVEC) layer. The confluent endothelium is visualized
using immunohistochemistry to stain for vascular endothelial
cadherin (VE-Cad), an endothelial-specific junction protein
expressed when cells form confluent networks (shown in
magenta).
[0139] To further promote the attachment and proliferation of the
endothelial cells along the fabricated channel walls, the interior
of the walls may be coated by perfusing a fibronectin solution
through the channels prior to introducing the HUVEC suspension, as
shown in FIGS. 9A-9D. Also, when animal blood is directly injected
into the inlet of the 2-D vascular network it rapidly flows through
the entire network to outlet. These initial demonstrations
illustrate the potential to create perfusable vasculature of nearly
any arbitrary design.
Printing of Cell-Laden Filaments Including More than One Cell
Type
[0140] The printing of cell co-culture inks that allow the delivery
of two or more cell types within a single ink filament is
demonstrated. It is observed that a cell-laden ink including, in
this example, a dispersion of HNDFs and HUVECs in a gelatin-fibrin
matrix material, leads to spontaneous neovasculature formation in
the printed filament, as evidenced in FIGS. 20A-20C. This is not
observed in printed filaments based on a monoculture of HUVECs.
This approach suggests cell-laden filaments comprising more than
one cell type, including tissue-specific cell types (e.g.,
hepatocytes, islets, podocytes, neurons, etc.) or stem cells (e.g.,
iPSCs, MSCs, etc), may be used to achieve desired heterogeneity and
also to enhance function.
Printing of Fugitive Ink onto a Cell-Laden Matrix
[0141] As in the previous example, HNDFs and HUVECs are dispersed
within an extracellular matrix composition (specifically, a
gelatin-fibrin matrix material) to form a cell-laden matrix. A
fugitive ink is printed directly onto the cell-laden matrix and
then encapsulated by the gelatin-fibrin matrix material. The
fugitive ink is evacuated to form vascular channels, and the
vascular channels are seeded with HUVECs. Over time, it is found
that the HUVECs assemble into capillary structures within the
printed cell-laden filament. FIGS. 19A-19D show that the
endothelial cells become attached to the vascular channels and form
confluent layers, and FIGS. 19E-19F show evidence of angiogenic
sprouting of small capillaries from the confluent blood vessels
indicating that the process is conducive to cellular remodeling and
higher-level biological processes.
[0142] Two effects are hypothesized to contribute to this observed
behavior. First, fibroblasts have been shown numerous times to be
pro-angiogenic support cells in vitro through specific chemical
cues such as fibroblast growth factor (FGF), often leading to
neovascularization processes. Additionally, the concentrated
population of proliferative cells within the matrix has extensive
metabolic requirements that are likely not met by diffusion alone.
It is widely accepted that cells that are not within a few 100
microns of blood vessel will become oxygen stressed and eventually
necrotic. In vivo, the recruitment of host vasculature into
avascular structures to prevent necrosis has been observed.
Printing of Tissue Constructs Including Interpenetrating
Vasculature
[0143] To demonstrate the fabrication of tissue constructs replete
with blood vessels, multiple types of cells, and an extracellular
matrix composition, 3D heterogeneous structures of varying design
are printed.
[0144] The first structure is composed of semi-woven features
printed in and out of plane (FIGS. 10A-10G). This four-layer tissue
construct includes two tissue patterns each comprising a different
cell type and a vascular pattern formed from sacrificial filaments
comprising a fugitive ink. The tissue construct is produced in a
layer by layer build sequence by printing four inks: PDMS, fugitive
Pluronic F127 and two different cell-laden GelMA inks, followed by
depositing pure GelMA ink at 37.degree. C. to fully encapsulate the
printed features, and finally photopolymerization to cross-link the
GelMA matrix. This 3D architecture was conceived and fabricated to
demonstrate the printing capabilities and also facilitate confocal
imaging through the entire 4-layer, printed construct.
[0145] As indicated previously, the PDMS ink is first printed in
the form of a high-aspect ratio border that surrounds each tissue
construct and serves as a mold for the pure GelMA ink used for the
encapsulation step. The fugitive ink and both cell-laden GelMA
inks, which contain either green fluorescent protein expressing
human neonatal dermal fibroblasts (HNDFs) or non-fluorescent
10T1/2s, an established mouse fibroblast line, are co-printed at
concentrations of 2.times.10.sup.6 cells/mL through 200 .mu.m
nozzles in a predefined sequential process. FIG. 10C shows an image
of the 3D structure directly after printing. Only the green
fluorescent channel is overlaid onto the bright field image so that
the printed cell channel can be easily visualized. After
fabrication, the fugitive ink is liquefied and removed from the 3D
construct. The evacuation procedure involves placing empty syringe
tips into the inlet and outlet microchannels and then suctioning
out the entire vascular network under a modest vacuum. The removal
process is rapid and yields a high fidelity, interpenetrating
vasculature, which is then endothelialized as described above.
Characterization of the Tissue Constructs
[0146] Using microscopy, the locations of the three cell types that
are independently stained (green-GFP HNDFs, blue-10T1/2, and
red-HUVECs) are identified. The semi-woven nature of this
engineered tissue construct is clearly visible in the schematics
and images shown in FIGS. 10B-10D. Using confocal laser scanning
microscopy, it is possible to fully interrogate this 3D tissue
construct and determine the precise locations of each cell.
Confocal microscopy images of the 3-D printed structure in XY, XZ,
and YZ after 2 days of culture are shown in FIG. 10G. To
demonstrate the versatility of this approach, other 3D tissue
construct were also designed and printed. Although it is difficult
to obtain confocal images due the dense, interpenetrating nature of
the cell-laden filaments, both the green fluorescent protein
expressing HNDFs in GelMA and the red-HUVECs that line the 3-D
vasculature network are visible.
Investigation of Cell Viability
[0147] As a final step, the viability of the printed 10T1/2
fibroblast cells over the course of one week was investigated. At
Day 0, the cell viability was 61%; however, it increased to 82%
after 7 days. While there is lower initial cell viability compared
with the control (78% on Day 0), the printed cells do proliferate
and spread over time leading to similar levels of viability after 1
week in culture. The decreased initial viability could arise from
the shear or extensional stress experienced by the cells during the
printing process. Applied pressure, nozzle diameter, cell type, and
environmental conditions may affect cell viability after printing.
Another critical parameter is the total build time required to
print the desired engineered tissue construct. There may be a
maximum time over which the cell-laden inks can be stored in the
ink reservoir prior to being harmed. However, implementation of
multinozzle print heads that were reported previously (J. A. Lewis
et al., "Multinozzle Deposition System for Direct Write
Applications," International Patent Application No.
PCT/US20121044794, filed Jun. 29, 2012, which is hereby
incorporated by reference) for high-throughput, multimaterial
printing, may reduce the characteristic build times by two orders
of magnitude in comparison with single nozzle printing. For
example, printing an engineered tissue construct with a volume of
1000 cm.sup.3, comparable to a typical adult human liver, could
require approximately 72 h using a single 200 .mu.m nozzle at
typical printing speeds. However, implementation of a
64-multinozzle array may reduce the respective build time to about
1 h.
[0148] A new approach has been developed and described in the
present disclosure for creating vascularized, heterogeneous tissue
constructs on demand via 3D bioprinting. This highly scalable
platform enables the fabrication of engineered tissue constructs in
which vasculature, multiple cell types and optionally other
functional chemical substances, such as drugs, toxins, proteins
and/or hormones, are programmably placed at desired locations
within an extracellular matrix. This technique may lead to the
rapid manufacturing of functional 3D tissues and organs needed for
transplant.
[0149] Although the present invention has been described in
considerable detail with reference to certain embodiments thereof,
other embodiments are possible without departing from the present
invention. The spirit and scope of the appended claims should not
be limited, therefore, to the description of the preferred
embodiments contained herein. All embodiments that come within the
meaning of the claims, either literally or by equivalence, are
intended to be embraced therein.
[0150] Furthermore, the advantages described above are not
necessarily the only advantages of the invention, and it is not
necessarily expected that all of the described advantages will be
achieved with every embodiment of the invention.
* * * * *