U.S. patent application number 16/029919 was filed with the patent office on 2018-11-01 for engineered tissues for in vitro research uses, arrays thereof, and methods of making the same.
The applicant listed for this patent is Organovo, Inc.. Invention is credited to Scott DORFMAN, Chirag KHATIWALA, Keith MURPHY, Sharon PRESNELL, Justin ROBBINS, Benjamin SHEPHERD.
Application Number | 20180313822 16/029919 |
Document ID | / |
Family ID | 47883950 |
Filed Date | 2018-11-01 |
United States Patent
Application |
20180313822 |
Kind Code |
A1 |
MURPHY; Keith ; et
al. |
November 1, 2018 |
Engineered Tissues for in vitro Research Uses, Arrays Thereof, and
Methods of Making the Same
Abstract
Disclosed are living, three-dimensional tissue constructs for in
vitro scientific and medical research, arrays thereof, and methods
of making said tissues and arrays.
Inventors: |
MURPHY; Keith; (Palos Verdes
Estates, CA) ; KHATIWALA; Chirag; (San Diego, CA)
; DORFMAN; Scott; (San Diego, CA) ; SHEPHERD;
Benjamin; (San Diego, CA) ; PRESNELL; Sharon;
(San Diego, CA) ; ROBBINS; Justin; (La Jolla,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Organovo, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
47883950 |
Appl. No.: |
16/029919 |
Filed: |
July 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13612768 |
Sep 12, 2012 |
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16029919 |
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61533757 |
Sep 12, 2011 |
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61533753 |
Sep 12, 2011 |
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61533761 |
Sep 12, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/50 20130101;
C12N 2513/00 20130101; A61L 27/38 20130101; C12N 5/0691 20130101;
C12N 2502/27 20130101; G01N 33/5088 20130101; C12N 5/0697 20130101;
G01N 33/5082 20130101; A61L 27/3891 20130101; C12N 2502/28
20130101; A61L 27/34 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; A61L 27/50 20060101 A61L027/50; C12N 5/071 20060101
C12N005/071; A61L 27/34 20060101 A61L027/34; A61L 27/38 20060101
A61L027/38 |
Claims
1-52. (canceled)
53. An in vitro living, three-dimensional tissue construct
comprising: (a) at least one adherent cell type; (b) a vascular or
pseudo vascular network and cells that are not part of the vascular
or pseudo vascular network; and (c) a multi-layered architecture,
wherein each layer of the multi-layered architecture is in direct
contact with at least one other layer, and wherein the layers are
cohered to form the construct; wherein the tissue construct is free
of pre-formed scaffold at the time of bioprinting and at the time
of use in an in vitro assay.
54. The tissue construct of claim 53, wherein the tissue construct
comprises at least one layer comprising a plurality of cell types
arranged relative to each other to create a planar geometry.
55. The tissue construct of claim 53, wherein the tissue construct
comprises at least one layer that is compositionally or
architecturally distinct from at least one other layer to create a
laminar geometry.
56. The tissue construct of claim 53, further comprising a
non-adherent cell type.
57. The tissue construct of claim 53, wherein the tissue construct
is at least about 25 .mu.m in its smallest dimension at the time of
bioprinting, wherein the tissue construct is no greater than about
3 cm in its largest dimension at the time of bioprinting, or
both.
58. The tissue construct of claim 53, wherein the at least one
adherent cell type is a non-differentiated cell.
59. The tissue construct of claim 53, wherein the at least one
adherent cell type originated from a tissue selected from the group
consisting of: liver, gastrointestinal, pancreatic, kidney, lung,
tracheal, vascular, skeletal muscle, cardiac, skin, smooth muscle,
connective tissue, corneal, genitourinary, breast, reproductive,
endothelial, epithelial, fibroblast, neural, Schwann, adipose,
bone, bone marrow, cartilage, pericytes, mesothelial, endocrine,
stromal, lymph, blood, endoderm, ectoderm, and mesoderm.
60. The tissue construct of claim 53, wherein the tissue construct
is a vascular wall segment.
61. An array of living, three-dimensional tissue constructs,
wherein each tissue construct in the array is a tissue construct of
claim 53.
62. A method of producing a living, three-dimensional tissue
construct for in vitro use comprising: a. bioprinting multiple
layers by extrusion of at least one semi-solid or solid bio-ink
comprising at least one adherent cell type, wherein at least one
semi-solid or solid bio-ink comprises endothelial cells, and
wherein each layer is in direct contact with at least one other
layer; and b. cohering the layers to form a living,
three-dimensional tissue construct comprising a vascular or pseudo
vascular network and cells that are not part of the vascular or
pseudo vascular network; wherein the tissue construct is free of
pre-formed scaffold at the time of bioprinting and at the time of
use in an in vitro assay.
63. The method of claim 62, wherein the bio-ink comprises
multicellular aggregates comprising mammalian cells.
64. The method of claim 62, wherein the bioprinting further
comprises arranging a plurality of cell types relative to each
other to create a planar geometry in at least one layer.
65. The method of claim 62, comprising bioprinting at least one
layer that is compositionally or architecturally distinct from at
least one other layer to create a laminar geometry.
66. The method of claim 62, wherein the tissue construct is at
least about 25 .mu.m in its smallest dimension at the time of
bioprinting, wherein the tissue construct is no greater than about
3 cm in its largest dimension at the time of bioprinting, or
both.
67. The method of claim 62, wherein the at least one adherent cell
type, the endothelial cells, or both are differentiated cells.
68. The method of claim 62, wherein the at least one adherent cell
type, the endothelial cells, or both are non-differentiated
cells.
69. The method of claim 62, comprising isolating the at least one
adherent cell type from a tissue selected from the group consisting
of: liver, gastrointestinal, pancreatic, kidney, lung, tracheal,
vascular, skeletal muscle, cardiac, skin, smooth muscle, connective
tissue, corneal, genitourinary, breast, reproductive, endothelial,
epithelial, fibroblast, neural, Schwann, adipose, bone, bone
marrow, cartilage, pericytes, mesothelial, endocrine, stromal,
lymph, blood, endoderm, ectoderm, mesoderm.
70. The method of claim 62, comprising isolating the endothelial
cells from a tissue selected from the group consisting of: blood,
blood vessel, lymphatic vessel, tissue of the digestive tract,
tissue of the genitourinary tract, adipose tissue, tissue of the
respiratory tract, tissue of the reproductive system,
mesoderm-derived tissue, bone marrow, and umbilical tissue.
71. The method of claim 62, wherein the tissue construct is a
vascular wall segment.
72. The method of claim 62, comprising producing multiple tissue
constructs and constructing an array from the multiple tissue
constructs.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/612,768, filed Sep. 12, 2012, which claims the benefit of
U.S. Application Ser. No. 61/533,757, filed Sep. 12, 2011, U.S.
Application Ser. No. 61/533,753, filed Sep. 12, 2011, and U.S.
Application Ser. No. 61/533,761, filed Sep. 12, 2011, all of which
are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] The research and development cost of a new pharmaceutical
compound is approximately $1.8 billion. See Paul, et al. (2010).
How to improve R&D productivity: the pharmaceutical industry's
grand challenge. Nature Reviews Drug Discovery 9(3):203-214. Drug
discovery is the process by which drugs are discovered and/or
designed. The process of drug discovery generally involves at least
the steps of: identification of candidates, synthesis,
characterization, screening, and assays for therapeutic efficacy.
Despite advances in technology and understanding of biological
systems, drug discovery is still a lengthy, expensive, and
inefficient process with low rate of new therapeutic discovery.
SUMMARY OF THE INVENTION
[0003] There is a need for materials, tools, and techniques that
substantially increase the number and quality of innovative,
cost-effective new medicines, without incurring unsustainable
R&D costs. Accordingly, the inventors describe herein
engineered mammalian tissues and vascular wall segments, arrays
thereof, and methods of making the same that have utility in tissue
and organ engineering, in vitro assays, drug discovery, and other
areas.
[0004] In one aspect, disclosed herein are living,
three-dimensional tissue constructs comprising: at least one
adherent cell type, the at least one adherent cell type cohered and
fused to form a living, three-dimensional tissue construct, the
tissue construct having a multi-layered architecture which is not a
vascular tube, the tissue construct for in vitro use, provided that
at least one component of the tissue construct was bioprinted. In
some embodiments, the tissue construct is substantially free of any
pre-formed scaffold at the time of bioprinting or at the time of
use. In some embodiments, the tissue construct comprises at least
one layer comprising a plurality of cell types, the cell types
spatially arranged relative to each other to create a planar
geometry. In some embodiments, the tissue construct comprises a
plurality of layers, at least one layer compositionally or
architecturally distinct from at least one other layer to create a
laminar geometry. In some embodiments, the tissue construct further
comprises non-adherent cell types. In some embodiments, the tissue
construct is secured to a biocompatible surface. In further
embodiments, the biocompatible surface is a porous membrane. In
further embodiments, the biocompatible surface is coated with one
of or more of the following: a biocompatible hydrogel, a protein, a
chemical, a peptide, antibodies, or growth factors. In still
further embodiments, the tissue construct is subjected to shear
force, caused by fluid flow, on one or more sides. In some
embodiments, the tissue construct is at least about 25 .mu.m in its
smallest dimension at the time of bioprinting. In some embodiments,
the tissue construct is no greater than about 3 cm in its largest
dimension at the time of bioprinting. In some embodiments, the
tissue construct is for use in in vitro assays. In further
embodiments, the tissue construct is for use in drug testing. In
some embodiments, the adherent cells are differentiated cells. In
other embodiments, the adherent cells are non-differentiated cells.
In some embodiments, the adherent cells originated from a tissue
selected from the group consisting of: liver, gastrointestinal,
pancreatic, kidney, lung, tracheal, vascular, skeletal muscle,
cardiac, skin, smooth muscle, connective tissue, corneal,
genitourinary, breast, reproductive, endothelial, epithelial,
fibroblast, neural, Schwann, adipose, bone, bone marrow, cartilage,
pericytes, mesothelial, endocrine, stromal, lymph, blood, endoderm,
ectoderm, and mesoderm. In some embodiments, the tissue construct
is a vascular wall segment.
[0005] In another aspect, disclosed herein are arrays of living,
three-dimensional tissue constructs, each tissue construct
comprising: at least one adherent cell type, the at least one
adherent cell type cohered and fused to form a living,
three-dimensional tissue construct, each tissue construct having a
multi-layered architecture, each tissue construct for in vitro use,
provided that at least one component of each tissue construct was
bioprinted. In some embodiments, each tissue construct is
substantially free of any pre-formed scaffold at the time of
bioprinting or the time of use. In some embodiments, the adherent
cells are selected from the group consisting of: liver cells,
gastrointestinal cells, pancreatic cells, kidney cells, lung cells,
tracheal cells, vascular cells, skeletal muscle cells, cardiac
cells, skin cells, smooth muscle cells, connective tissue cells,
corneal cells, genitourinary cells, breast cells, reproductive
cells, endothelial cells, epithelial cells, fibroblast, neural
cells, Schwann cells, adipose cells, bone cells, bone marrow cells,
cartilage cells, pericytes, mesothelial cells, cells derived from
endocrine tissue, stromal cells, stem cells, progenitor cells,
lymph cells, blood cells, endoderm-derived cells, ectoderm-derived
cells, mesoderm-derived cells, and combinations thereof. In some
embodiments, each tissue construct within the array is
substantially similar. In other embodiments, one or more of the
tissue constructs within the array is unique. In some embodiments,
one or more individual tissues within the array represent human
tissues selected from the group consisting of: blood or lymph
vessel, muscle, uterus, nerve, mucous membrane, mesothelium,
omentum, cornea, skin, liver, kidney, heart, trachea, lung, bone,
bone marrow, adipose, connective, bladder, breast, pancreas,
spleen, brain, esophagus, stomach, intestine, colon, rectum, ovary,
prostate, endocrine tissue, endoderm, mesoderm, and ectoderm. In
some embodiments, each tissue construct exists in a well of a
biocompatible multi-well container. In further embodiments, the
wells are coated with one of or more of the following: a
biocompatible hydrogel, a protein, a chemical, a peptide,
antibodies, or growth factors. In further embodiments, each tissue
construct was placed onto a porous, biocompatible membrane within
the wells of the container. In further embodiments, the container
is compatible with an automated or semi-automated drug screening
process. In some embodiments, each tissue construct is secured to a
biocompatible surface. In further embodiments, the biocompatible
surface is a porous membrane. In further embodiments, the
biocompatible surface is coated with one of or more of the
following: a biocompatible hydrogel, a protein, a chemical, a
peptide, antibodies, or growth factors. In still further
embodiments, each tissue construct is subjected to shear force,
caused by fluid flow, on one or more sides. In some embodiments,
each tissue construct within the array is maintained independently
in culture. In other embodiments, two or more individual tissue
constructs within the array exchange soluble factors. In some
embodiments, the array is for use in in vitro assays. In further
embodiments, the array is for use in drug testing. In some
embodiments, at least one tissue within the array is a vascular
wall segment.
[0006] In another aspect, disclosed herein are living,
three-dimensional tissue constructs comprising: one or more layers,
wherein each layer contains one or more cell types, the one or more
layers cohered to form a living, three-dimensional tissue
construct, the tissue construct characterized by having at least
one of: at least one layer comprising a plurality of cell types,
the cell types spatially arranged relative to each other to create
a planar geometry; and a plurality of layers, at least one layer
compositionally or architecturally distinct from at least one other
layer to create a laminar geometry. In some embodiments, at least
one component of the tissue construct was bioprinted. In further
embodiments, the tissue construct is substantially free of any
pre-formed scaffold at the time of bioprinting or at the time of
use. In some embodiments, the tissue construct is for use in in
vitro assays. In further embodiments, the tissue construct is for
use in drug testing.
[0007] In another aspect, disclosed herein are methods for
constructing a living, three-dimensional tissue construct
comprising the steps of: bioprinting bio-ink comprising at least
one adherent cell type into or onto a form; and fusing of the
bio-ink into a living, three-dimensional tissue construct; provided
that the tissue construct is for in vitro use and not a vascular
tube. In some embodiments, the tissue construct is free of any
pre-formed scaffold at the time of bioprinting or the time of use.
In some embodiments, the form is bioprinted. In further
embodiments, the form is bioprinted substantially contemporaneously
with the bio-ink. In some embodiments, the method further comprises
the step of dissolving the form.
[0008] In another aspect, disclosed herein are methods of
constructing a living, three-dimensional tissue construct
comprising the steps of: preparing one or more cohered
multicellular aggregates comprising mammalian cells; placing said
one or more cohered multicellular aggregates onto a support to form
at least one of: at least one layer comprising a plurality of cell
types, the cell types spatially arranged relative to each other to
create a planar geometry; and a plurality of layers, at least one
layer compositionally or architecturally distinct from at least one
other layer to create a laminar geometry; and incubating said one
or more multicellular aggregates to allow them to cohere and to
form a living, three-dimensional tissue construct. In some
embodiments, at least one component of the tissue construct was
bioprinted. In further embodiments, the tissue construct is free of
any pre-formed scaffold at the time of bioprinting or the time of
use.
[0009] In another aspect, disclosed herein are methods of
constructing an array of living, three-dimensional tissue
constructs comprising the steps of: preparing cohered multicellular
aggregates comprising mammalian cells; placing said cohered
multicellular aggregates onto a biocompatible support; wherein said
aggregates are spatially arranged in a form suitable for a tissue
array; and incubating said multicellular aggregates to allow them
to cohere and form an array of living, three-dimensional tissue
constructs. In some embodiments, at least one component of each
tissue construct was bioprinted. In further embodiments, each
tissue construct is substantially free of any pre-formed scaffold
at the time of bioprinting or the time of use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0011] FIG. 1 depicts non-limiting examples of bioprinted vascular
wall segments constructed with polytypic SMC:EC bio-ink in
cylindrical format. Various staining conditions are shown to
indicate distribution and position of cell types. (L to R)
Bioprinted vessel wall constructs immediately after bioprinting in
a 6-well plate. Hematoxylin and Eosin (H&E) staining of a
construct after 5 days in culture demonstrating fusion of
individual bio-ink particles into a contiguous structure and
organization of cells at the periphery. CD31 staining of constructs
generated with multicellular SMC:EC bio-ink shows organization of
CD31-positive EC at the periphery and scattered CD31-positive cells
within the wall. Trichrome staining of vessel wall constructs after
5 days shows robust collagen formation.
[0012] FIG. 2a is a macroscopic image depicting a non-limiting
example of three-dimensional bioprinted vascular wall segments 24
hours post printing. The patch was constructed with polytypic
bio-ink cylinders of multicellular HASMC:HAEC at a ratio of
85:15.
[0013] FIG. 2b depicts histology images of bioprinted patches
non-limiting examples of bioprinted vascular wall segments
constructed with bio-ink comprised of multicellular HASMC:HAEC at a
ratio of 85:15. HAEC stain positive for CD31.
[0014] FIG. 3 depicts non-limiting examples of bioprinted vascular
wall segments constructed with SMC-only bio-ink cylinders followed
by bioprinting of a second layer composed of EC concentrate,
creating a laminar architecture. Various staining conditions are
shown to indicate distribution and position of cell types. (L to R)
H&E, CD31, a-SMA and TUNEL staining of vessel wall constructs
bioprinted with SMC bio-ink to form a first layer atop a porous
membrane, followed by deposition of an EC concentrate from the
NovoGen MMX Bioprinter.TM. to form a second layer. Following 5 days
of culture organization of an EC lining is observed on the top of
the construct and an SMC rich vessel construct wall is present. A
limited number of TUNEL-positive nuclei are found throughout the
bioprinted structure.
[0015] FIG. 4a depicts non-limiting examples of bioprinted vascular
wall segments constructed with HASMC bioprinted on top of a first
layer of human dermal fibroblasts (HDFa) and subsequently layered
with HAEC, creating a tri-layered laminar architecture. Depicted
are histology images of tri-layered bioprinted patches. Patch made
using HASMC bio-ink printed on top of a confluent layer of HDFa on
a Transwell.RTM. membrane, and finally top seeded with HAEC to form
a third layer. HAEC cells stain positive for CD31. HASMC stain
positive for alpha SMA. Timepoint=4 days post printing.
[0016] FIG. 4b is a macroscopic image depicting a non-limiting
example of HASMC bio-ink bioprinted within a co-printed NovoGel.TM.
containment window and layered with HAEC, but without a third layer
of NovoGel.TM. lattice (e.g., mesh) on top. Depicted is a
macroscopic image of three-dimensional bioprinted patch. Shown is a
2.times. magnification image of cylindrical HASMC bio-ink shown
immediately after bioprinting. HAECs were bioprinted on top of the
HASMC patch. A top layer of NovoGel.TM. mesh was not utilized on
this construct.
[0017] FIG. 5 is a non-limiting example of a bioprinted cell sheet
and a temporary or removable bioprinted confinement lattice
structure; also depicted are exemplary steps for fabricating the
same. A simple example of a lattice structure printed on the top
surface of a three-dimensional cell sheet. (A) Optionally
dispensing base layer of confinement material. (B) Optionally
dispensing a perimeter of confinement material. (C) Bioprinting
cells within a defined geometry. (D) Dispensing cylinders of
confinement material overlaying the cells.
[0018] FIG. 6 is a macroscopic image depicting a non-limiting
example of an engineered liver tissue, in this case, a
multi-layered liver tissue bioprinted using a continuous deposition
mechanism using bio-ink composed of multiple liver cell types
encapsulated in a water-soluble extrusion compound (e.g., PF-127).
(A) shows a schematic diagram of a single functional unit
highlighting the planar geometry created by patterning bio-ink and
negative space; (B) shows tessellated, bioprinted functional units
bioprinted with PF-127 containing 2.times.10.sup.8 cells; (C) and
(D) show the construct after application of media and dissolution
of the extrusion compound, 20 minutes and 18 hours after
application of media to the structure, respectively; note retention
of the planar geometry over time.
[0019] FIG. 7 is a photomicrograph of the H&E stained
tessellated construct of FIG. 6, depicting an exemplary "spoke" in
the tessellated construct. Shown is H&E staining of
formalin-fixed paraffin-embedded tissue sections of stellate cells,
endothelial cells, and dermal fibroblasts bioprinted by continuous
deposition in a multi-layer tessellated hexagonal structure and
then cultured for 18 hours.
[0020] FIG. 8 is a non-limiting schematic diagram (A), macroscopic
photograph (B), and series of photomicrographs (C-E) of a
bioprinted neotissue with laminar geometry. A NovoGel.TM. hydrogel
base and co-printed confining box were bioprinted, followed by
deposition of a first layer comprising liver epithelial cell
bio-ink (HepG2 cells), onto which a second layer was bioprinted
comprised of hepatic stellate cells and endothelial cells. In this
example, the stellate:EC layer was bioprinted via continuous
deposition of bio-ink containing a hydrogel extrusion compound (A).
Gross images of construct immediately after fabrication
demonstrating the two distinct layers of bio-ink (B). H&E
staining of sections of formalin-fixed paraffin-embedded constructs
(C) following 48 hours of culture reveals distinct morphology of
the two layers and establishment of a laminar geometry.
CD31-positive cells are restricted to the upper layer of the
construct where a suspension of endothelial cells and hepatic
stellate cells were bioprinted (D), while IGF-2-positive HepG2 are
found only in the bottom layer (E).
[0021] FIG. 9 is a series of photomicrographs depicting cell
patterning and layering in bioprinted tissues. H&E staining of
paraffin-embedded tissue sections reveals a contiguous neotissue
(A) formed by bioprinting polytypic cell populations containing
vascular endothelial cells. Staining of the tissue sections with
antibody directed at CD31 reveals the presence of centrally-located
EC-lined microvessels and an external layer of CD31-positive EC
(B). Forced layering was also done by bioprinting a continuous
sheet of vascular SMC bio-ink (C) and bioprinting an external layer
of CD31-positive EC (D).
[0022] FIG. 10 is a non-limiting schematic diagram of a bioprinted
human lung tissue construct with laminar geometry, depicting steps
for fabrication. A double-walled box using hydrogel cylinders is
bioprinted on the cell culture insert membrane (A). Next, NHLF:EC
bio-ink cylinder is then bioprinted inside the box (B). The SAEC
suspension is then bioprinted on top of the NHLF-EC tissue (C). The
bioprinted lung tissue construct is constrained with a top layer of
hydrogel cylinders (D) and the construct submerged in complete
media for culture.
[0023] FIG. 11 is a series of photomicrographs depicting
characterization of bioprinted human lung tissues. H&E staining
of formalin-fixed tissue sections from bioprinted lung tissue after
12 d in culture reveals tissue fusion (A). CD31-positive EC are
found organized throughout (B) and .alpha.-SMA-positive NHLF (C)
localized at the periphery of the construct. Cytokeratin
19-positive SAEC are found only at the apical surface of the tissue
(D). Stimulation with 10 ng/mL IL-13 results in thickening of the
tissue (E) and increased organization of CD31-positive EC within
the construct wall (F). Cytokine stimulation also increases the
number of a-SMA positive NHLF found in the sub-epithelial zone (G).
CK19-positive SAEC remain confined to the apical surface (H).
[0024] FIG. 12 is a pair of macroscopic photographs depicting a
bioprinted vascular wall segment immediately after bioprinting (A)
and following 24 hours of incubation in media (B). Bi-layered blood
vessel wall segments were bioprinted with SMC or SMC:Fb bio-ink and
highly-concentrated EC cell suspensions. Immediately after
bioprinting (A) individual bio-ink cylinders are identifiable.
Following 24 hours of incubation in media, the individual bio-ink
cylinders and layer of EC have completely fused to form a single
contiguous construct (B).
[0025] FIG. 13 is a series of photomicrographs depicting analysis
of multi-layered blood vessel wall segments with laminar geometry
bioprinted on multi-well cell culture inserts under static and flow
conditions. H&E staining of formalin-fixed paraffin-embedded
tissues reveals well formed tissue constructs under both static (A)
and flow/shear stress (D) culture conditions. Static culture of
blood vessel constructs was sufficient to maintain proper cell
arrangement which was characterized by a layer of CD31-positive EC
at the laminar surface (C) and an .alpha.-SMA-positive SCM-rich
media. Following exposure to 5 mL/min flow in a flow cell chamber,
the CD31-positive EC layer appeared thicker (E) and the
.alpha.-SMA-positive SMC-rich media appears to also be thicker and
more well-organized, suggestive of a positive response to the
biomechanical stimuli associated with shear stress and fluid
flow.
[0026] FIG. 14 is a pair of non-limiting macroscopic photographs
depicting tissues bioprinted in multi-well plates (A) or within
multi-well cell culture inserts (B). Bioprinted tissue constructs
are generated in multi-well plates (A) or within multi-well culture
inserts (B), which are optionally placed in an appropriate
multi-well plate for long-term maintenance and maturation. Here,
tissue constructs were bioprinted in a 24-well polystyrene plate
(A) and on the porous membrane of a 6-well cell culture insert
(B).
[0027] FIG. 15 is a pair of non-limiting photomicrographs depicting
stimulation of bioprinted multi-layered blood vessel wall segments
with TGF-.beta.1. Stimulation of the bioprinted blood vessel wall
segment with the fibroproliferative cytokine TGF-.beta.1 (10 ng/mL)
results in a significant increase in collagen deposition and
organization as seen by trichrome staining of formalin-fixed
paraffin-embedded tissue constructs following 5+ days of
stimulation. Control (A), TGF-.beta.1-treated (B).
[0028] FIG. 16 is a series of photomicrographs depicting
stimulation of bioprinted liver tissue containing hepatic stellate
cells with TGF-.beta.1. Incubation of bioprinted hepatic stellate
cell sheets with increasing concentrations of TGF-b1 (0, 1, 10, 50
ng/mL), results in changes in gross observation of the bioprinted
tissues as increases in cytokine concentration lead to increases in
tissue outgrowth formation (A-D, 0-50 ng/mL). Trichrome staining of
tissue sections from bioprinted hepatic stellate-containing tissues
reveals increases in collagen deposition and construct size and
dramatic decreases in cell density (E-H, 0-50 ng/mL).
[0029] FIG. 17 is a pair of macroscopic photographs depicting
co-molded functional liver tissue microstructure formed by
continuous deposition bioprinting of a patterned 6-layer hexagon of
PF-127 with bioprinting of cell paste into each triangle (A),
followed by dissolution of PF-127 border (B). Dissolution of PF-127
border after media addition allows for distinct regions to be
created and additional cells types and complexity to be generated
(B).
[0030] FIG. 18 is a series of non-limiting examples of planar (A-C)
and laminar (D-E) geometries, including combinations thereof (F)
that are compatible with the methods of construction described
herein, and reproduce architectural or spatial elements of native
tissue architecture and biology.
[0031] FIG. 18A shows schematic diagrams of planar geometry
examples (top view) of bioprinted tissues: (1) glandular
tissue/cancer tissue, comprising bio-ink #1 (e.g., epithelial),
bio-ink #2 (e.g., stromal) and a vascular component; (2) composite
tissue/tissue interface, comprising bio-ink #1 (e.g., cartilage),
bio-ink #2 (e.g., bone) and a third component (e.g., bone marrow);
(3) architecturally-correct tissue with a vascular network,
comprising bio-ink #1 (e.g., parenchymal tissue, including for
example liver, pancreas, adipose, renal, muscle, skin, bone,
cartilage, nervous, neural, urologic, cardiovascular, lymphoid,
ocular, aural, or endocrine tissues, in any planar pattern) and
bio-ink #2 (e.g., vascular network), with an optional flow through
the bioprinted tissue; (4) zonal tissues, comprising bio-ink #1
(e.g., renal cortex), bio-ink #2 (e.g., renal medulla) and bio-ink
#3 (e.g., renal papilla). The interface between bio-ink #1 and
bio-ink #2 represents the cortico-medullary junction, for example;
and (5) lobulated tissues, comprising bio-ink #1 (e.g., liver
lobules), bio-ink #2 (e.g., stromal/vascular tissue). Note that
each geometric "lobule" may also have spatially-directed
architecture within it.
[0032] FIG. 18B shows continued schematic diagrams of planar
geometry examples (top view) of bioprinted tissues: (6)
perfused/arrayed tissues, comprising component #1 (representing
channels (i.e., architected to exist as void spaces), vessels
(i.e., artery, vein, lymph) or tubes with lumens (i.e., ducts,
tubules generated from cells, and/or cell-material composites) and
component #2 (each patch can be same or different shape/size,
patches can be the same (multiples of the same tissue type),
patches can be distinct (multiple tissue types presented within the
interconnected grid); each patch may contain one or more cell types
and may have one or more architectural or geometrical spatial
pattern, achieved by directed patterning in the x, y, and/or z
plane; and each patch may be a composite of one or more tissue
types (e.g. bone and cartilage); (7) solid and liquid tissue/liquid
interfaces, comprising bio-ink #1 (the outer wall of a luminal
structure--blood vessel, heart, lymph vessel, stomach, bladder,
esophagus, intestine, bone, renal tubule, uterus, airway, fallopian
tube, etc.), bio-ink #2 (can be the inner wall of a luminal
structure when required--the vascular media, for example, or the
mucosal lining of a luminal component of the gastrointestinal
system, the epithelial or endothelial lining of a tubular
structure, for example), and a third component (a fluid, optionally
containing cells or biologically-relevant components (e.g.,
protein, drugs, pathogens etc.) that interact with the lumenal
structure wall as a lumenal fluid or cell-containing solution. The
fluid is a liquid or semi-liquid component with a + or - flow
through the tissue. In some embodiments, a lining of cells
(endothelial, epithelial) may be present in the single- or
double-walled structure to serve as the physiologically-correct
barrier. In some embodiments, the interactions between component #3
and lumenal surface/wall tissue can be observed by ensuring that
the top and/or bottom surface(s) of the container are optically
clear.
[0033] FIG. 18C shows a continued schematic diagram of a planar
geometry example (top view) of a bioprinted tissue. (8)
endocrine/exocrine pancreas tissue, comprising endocrine
tissue/islets, exocrine tissue/pancreatic acinar cells and
supporting connective tissue. The tissue optionally contains an
incorporated microvascular network.
[0034] FIG. 18D shows schematic diagrams of laminar geometry
examples (side view) of bioprinted tissues. Barrier tissues and
specific examples of barrier-like tissues (airway, renal tubule and
intestine) are shown. (1) Exemplary barrier tissue, comprising a
barrier layer (endothelial or epithelial; single or multiple cells
types; one or more cell layers; cells may be positional patterned
(small.fwdarw.large airway), an interstitial layer/wall and/or
surface of a lumenal tissue, a porous mesh or membrane and an
optional endothelial layer. (2) Exemplary airway barrier-like
tissue, comprising airway epithelial cells from any level of the
airway and an interstitial layer with one or more of: fibroblasts,
smooth muscle cells, cartilaginous cells, incorporating a vascular
network comprising endothelial cells. Note that the interstitial
layer optionally includes planar geometry, for example, spatial
positioning of smooth muscle, fibroblasts, and cartilage components
as well as positioning of the endothelial network. (3) Exemplary
renal tubule barrier-like tissue, comprising renal tubular
epithelial cells (homogenous or heterogeneous and can optionally be
spatially arranged proximal tubule.fwdarw.collecting duct, for
example), an interstitial tissue layer (containing real stromal
cells, one or more of the following; vascular cells,
erythropoietin-producing cells, pericytes, mesenchymal cells,
glomerular cells; also comprising a vascular network); a porous
mesh or membrane support and an optional endothelial barrier. (4)
Exemplary intestinal barrier-like tissue, comprising an epithelial
layer, submucosa layer, and a muscularis layer comprising smooth
muscle cells. The epithelial layer comprises gut epithelium, with
the potential to utilize epithelial cells from various portion of
the gut tube, spatially positional cells to provide directionality
to the tissue (small.fwdarw.large intestine, for example). A
microvascular network is incorporated into submucosa/mucosa.
[0035] FIG. 18E shows continued schematic diagrams of laminar
geometry examples (side view) of bioprinted tissues. Specific
examples of barrier-like tissues are shown. (5) Exemplary mucosal
surface (e.g., oral) barrier-like tissue, comprising a mucosal
layer (comprising epithelial cells and one or more layers of cells,
optionally patterned) and an underlying submucosal layer, which may
comprise connective tissue, smooth muscle cell and optionally
comprise a microvascular network. The surfaces can be constructed
from oral, gut, nasal, bladder, bronchial, uterine (endometrial),
or penile mucosa. Composite constructs can also be made, a
mucocutaneous junction, for example, that adjoins skin to a mucosal
tissue such as intestine or bladder. (6) Exemplary mucocutaneous
junction barrier-like tissue, comprising an epithelial layer, a
lamina propria layer, and a smooth muscle layer on the mucosal side
(e.g., oral, nasal conjunctival, urethral, vaginal, anal) and an
epidermal layer, a dermal layer, and a skeletal muscle layer,
respectively, on the epithelium/skin side
(epithelium.fwdarw.epidermis, lamina propria.fwdarw.dermis, smooth
muscle.fwdarw.skeletal muscle). Additional tissue-specific
components (nerve, gland, etc) can be added in spatially defined
locations.
[0036] FIG. 18F shows schematic diagrams of combined planar and
laminar geometry examples of bioprinted tissues. (1) Exemplary
combined planar and laminar geometry bioprinted tissue (top view),
comprising a bio-ink #1 in any planar pattern (e.g. lobulated
pattern), which can be repeated precisely in the Z axis and a
bio-ink #2. (2) Cross-section of exemplary combined planar and
laminar geometry bioprinted tissue showing multiple layers (layer
1, layer 2, layer 3). The planar patterns are repeated in layers so
that thicker tissues are built up, carrying both the planar
geometry elements and the vertical (z-axis) continuations of the
pattern into the final tissue product. Features may include
contiguous channels, cellular compartments within a tissue (e.g.,
epithelial glands within a stromal field). Planar patterns may also
be varied in the z-axis, layer by layer, to create architectural
features. (3) Cross-section of exemplary combined planar and
laminar geometry bioprinted tissue showing multiple layers varied
in the z-axis. The diagram shows a cross-section of an example of
renal tissue comprising medullary renal tissue, papillary renal
tissue, and renal tubules, for example, in multiple layers. A
similar strategy can be applied to build specific features (glands,
follicles, tubes, dusts etc.) into multi-layered tissue
structures.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The invention relates to the field of regenerative medicine
and tissue and/or organ engineering. More particularly, the
invention relates to arrays of engineered mammalian tissues,
engineered vascular wall segments, arrays thereof, and methods of
fabrication.
[0038] Disclosed herein, in certain embodiments, are living,
three-dimensional tissue constructs comprising: at least one
adherent cell type, the at least one adherent cell type cohered and
fused to form a living, three-dimensional tissue construct, the
tissue construct having a multi-layered architecture which is not a
vascular tube, the tissue construct for in vitro use, provided that
at least one component of the tissue construct was bioprinted.
[0039] Also disclosed herein, in certain embodiments, are arrays of
living, three-dimensional tissue constructs, each tissue construct
comprising: at least one adherent cell type, the at least one
adherent cell type cohered and fused to form a living,
three-dimensional tissue construct, each tissue construct having a
multi-layered architecture, each tissue construct for in vitro use,
provided that at least one component of each tissue construct was
bioprinted.
[0040] Also disclosed herein, in certain embodiments, are living,
three-dimensional tissue constructs comprising: one or more layers,
wherein each layer contains one or more cell types, the one or more
layers cohered to form a living, three-dimensional tissue
construct, the tissue construct characterized by having at least
one of: at least one layer comprising a plurality of cell types,
the cell types spatially arranged relative to each other to create
a planar geometry; and a plurality of layers, at least one layer
compositionally or architecturally distinct from at least one other
layer to create a laminar geometry.
[0041] Also disclosed herein, in certain embodiments, are methods
for constructing a living, three-dimensional tissue construct
comprising the steps of: bioprinting bio-ink comprising at least
one adherent cell type into or onto a form; and fusing of the
bio-ink into a living, three-dimensional tissue construct; provided
that the tissue construct is for in vitro use and not a vascular
tube.
[0042] Also disclosed herein, in certain embodiments, are methods
of constructing a living, three-dimensional tissue construct
comprising the steps of: preparing one or more cohered
multicellular aggregates comprising mammalian cells; placing said
one or more cohered multicellular aggregates onto a support to form
at least one of: at least one layer comprising a plurality of cell
types, the cell types spatially arranged relative to each other to
create a planar geometry; and a plurality of layers, at least one
layer compositionally or architecturally distinct from at least one
other layer to create a laminar geometry; and incubating said one
or more multicellular aggregates to allow them to cohere and to
form a living, three-dimensional tissue construct.
[0043] Also disclosed herein, in certain embodiments, are methods
of constructing an array of living, three-dimensional tissue
constructs comprising the steps of: preparing cohered multicellular
aggregates comprising mammalian cells; placing said cohered
multicellular aggregates onto a biocompatible support; wherein said
aggregates are spatially arranged in a form suitable for a tissue
array; and incubating said multicellular aggregates to allow them
to cohere and form an array of living, three-dimensional tissue
constructs.
Certain Definitions
[0044] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0045] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "a nucleic acid" includes one or more nucleic acids,
and/or compositions of the type described herein which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth. Any reference to "or" herein is intended
to encompass "and/or" unless otherwise stated.
[0046] As used herein, "array" means a scientific tool including an
association of multiple elements spatially arranged to allow a
plurality of tests to be performed on a sample, one or more tests
to be performed on a plurality of samples, or both.
[0047] As used herein, "assay" means a procedure for testing or
measuring the presence or activity of a substance (e.g., a
chemical, molecule, biochemical, protein, hormone, or drug, etc.)
in an organic or biologic sample (e.g., cell aggregate, tissue,
organ, organism, etc.).
[0048] As used herein, "biocompatible" means posing limited risk of
injury or toxicity to cells. As presented in the specification and
claims, "biocompatible multi-well containers" and "biocompatible
membranes" pose limited risk of injury or toxicity to mammalian
cells, but the definition does not extend to imply that these
biocompatible elements could be implanted in vivo into a
mammal.
[0049] As used herein, "bioprinting" means utilizing
three-dimensional, precise deposition of cells (e.g., cell
solutions, cell-containing gels, cell suspensions, cell
concentrations, multicellular aggregates, multicellular bodies,
etc.) via methodology that is compatible with an automated or
semi-automated, computer-aided, three-dimensional prototyping
device (e.g., a bioprinter).
[0050] As used herein, "blood vessel" means a singular simple or
branched tubular structure having a smooth muscle cell-comprising
wall and endothelial cells lining the lumen, and having an internal
diameter greater than 100 .mu.m, and not existing as a component of
three-dimensional tissue construct that comprises non-blood vessel
tissue.
[0051] As used herein, "cohere," "cohered," and "cohesion" refer to
cell-cell adhesion properties that bind cells, cell aggregates,
multicellular aggregates, multicellular bodies, and/or layers
thereof. The terms are used interchangeably with "fuse," "fused,"
and "fusion."
[0052] As used herein, "laminar" means a multi-layered bioprinted
tissue in which two or more planar layers are combined to increase
the overall thickness of the tissue in the z-plane. In some
embodiments, each planar layer is substantially similar in
architecture and/or composition. In other embodiments, each planar
layer is substantially distinct in architecture and/or composition.
See, e.g., FIGS. 18A-F.
[0053] As used herein, "multi-layered" means being comprised of two
or more layers of tissue, wherein each tissue layer is one or more
cell-layers in thickness. In some embodiments, layers of tissue are
deposited one at a time. In other embodiments, multiple layers are
deposited simultaneously. Optionally, each layer is comprised of
multiple cell types. Further, the multiple cell types within each
layer are optionally arranged relative to each other in a
spatially-defined architecture in the x-y planes (i.e., horizontal
planes). Furthermore, addition of layers in the z-plane (i.e.,
vertical plane), in some cases, results in controlled spatial
positioning of the cells within the layers relative to each other
so that a spatially-defined architecture is continued in the
z-plane.
[0054] As used herein, "planar" means a layer of multicellular
bioprinted tissue in which multiple bio-ink compositions and/or
void spaces are spatially arranged into a defined pattern relative
to each other within the x-y plane of the tissue layer. See, e.g.,
FIGS. 18A-F.
[0055] As used herein, "scaffold" refers to synthetic scaffolds
such as polymer scaffolds and porous hydrogels, non-synthetic
scaffolds such as pre-formed extracellular matrix layers, dead cell
layers, and decellularized tissues, and any other type of
pre-formed scaffold that is integral to the physical structure of
the engineered tissue and/or organ and not able to be removed from
the tissue and/or organ without damage/destruction of said tissue
and/or organ. In further embodiments, decellularized tissue
scaffolds include decellularized native tissues or decellularized
cellular material generated by cultured cells in any manner; for
example, cell layers that are allowed to die or are decellularized,
leaving behind the ECM they produced while living. The term
"scaffoldless," therefore, is intended to imply that scaffold is
not an integral part of the engineered tissue at the time of use,
either having been removed or remaining as an inert component of
the engineered tissue. "Scaffoldless" is used interchangeably with
"scaffold-free" and "free of pre-formed scaffold."
[0056] As used herein, "subject" means any individual, which is a
human, a non-human animal, any mammal, or any vertebrate. The term
is interchangeable with "patient," "recipient" and "donor."
[0057] As used herein, "tissue" means an aggregate of cells.
Examples of tissues include, but are not limited to, connective
tissue (e.g., areolar connective tissue, dense connective tissue,
elastic tissue, reticular connective tissue, and adipose tissue),
muscle tissue (e.g., skeletal muscle, smooth muscle and cardiac
muscle), genitourinary tissue, gastrointestinal tissue, pulmonary
tissue, bone tissue, nervous tissue, and epithelial tissue (e.g.,
simple epithelium and stratified epithelium), endoderm-derived
tissue, mesoderm-derived tissue, and ectoderm-derived tissue.
Tissue Engineering
[0058] Tissue engineering is an interdisciplinary field that
applies and combines the principles of engineering and life
sciences toward the development of biological substitutes that
restore, maintain, or improve tissue function through augmentation,
repair, or replacement of an organ or tissue. The basic approach to
classical tissue engineering is to seed living cells into a
biocompatible and eventually biodegradable environment (e.g., a
scaffold), and then culture this construct in a bioreactor so that
the initial cell population expands further and matures to generate
the target tissue upon implantation. With an appropriate scaffold
that mimics the biological extracellular matrix (ECM), the
developing tissue, in some cases, adopts both the form and function
of the desired organ after in vitro and in vivo maturation.
However, achieving high enough cell density with a native
tissue-like architecture is challenging due to the limited ability
to control the distribution and spatial arrangement of the cells
throughout the scaffold. These limitations often result in tissues
or organs with poor mechanical properties and/or insufficient
function. Additional challenges exist with regard to biodegradation
of the scaffold, entrapment of residual polymer, and industrial
scale-up of manufacturing processes. Scaffoldless approaches have
been attempted. Current scaffoldless approaches are subject to
several limitations: [0059] Complex planar and/or laminar
geometries, such as multi-layered structures wherein one or more
layers is compositionally or architecturally distinct from other
layers or wherein one or more layers comprise multiple cell types
in spatially-defined positions relative to each other, often
require definitive, high-resolution placement of cell types within
a specific architecture to reproducibly achieve a native
tissue-like outcome. [0060] Scale and geometry are limited by
diffusion and/or the requirement for functional vascular networks
for nutrient supply. [0061] The viability of the tissues is, in
some cases, compromised by confinement material that limits
diffusion and restricts the cells' access to nutrients.
[0062] Disclosed herein, in certain embodiments, are engineered
mammalian tissues, engineered vascular wall segments, arrays
thereof, and methods of fabrication. The tissue engineering methods
disclosed herein have the following advantages: [0063] They are
capable of producing cell-comprising tissues and/or organs. [0064]
They mimic the environmental conditions found within the
development, homeostasis, and/or pathogenesis of natural tissues by
re-creating native tissue-like intercellular interactions. [0065]
They optionally achieve living, three-dimensional tissues and
compound tissues with a broad array of complex topologies and
geometries (e.g., multilayered structures, segments, sheets, tubes,
sacs, etc.). [0066] They are compatible with automated or
semi-automated means of manufacturing and are scalable.
[0067] Bioprinting enables improved methods of generating
micro-scale tissue analogues including those useful for in vitro
assays (see below).
Bioprinting
[0068] In some embodiments, at least one component of the
engineered tissues, including vascular wall segments, and arrays
thereof is bioprinted. In further embodiments, bioprinted
constructs are made with a method that utilizes a rapid prototyping
technology based on three-dimensional, automated, computer-aided
deposition of cells, including cell solutions, cell suspensions,
cell-comprising gels or pastes, cell concentrations, multicellular
bodies (e.g., cylinders, spheroids, ribbons, etc.), and,
optionally, confinement material onto a biocompatible surface
(e.g., composed of hydrogel and/or a porous membrane) by a
three-dimensional delivery device (e.g., a bioprinter). As used
herein, in some embodiments, the term "engineered," when used to
refer to tissues and/or organs means that cells, cell solutions,
cell suspensions, cell-comprising gels or pastes, cell
concentrates, multicellular aggregates, and layers thereof are
positioned to form three-dimensional structures by a computer-aided
device (e.g., a bioprinter) according to a computer script. In
further embodiments, the computer script is, for example, one or
more computer programs, computer applications, or computer modules.
In still further embodiments, three-dimensional tissue structures
form through the post-printing fusion of cells or multicellular
bodies which, in some cases, is similar to self-assembly phenomena
in early morphogenesis.
[0069] While a number of methods are available to arrange cells,
multicellular aggregates, and/or layers thereof on a biocompatible
surface to produce a three-dimensional structure including manual
placement, positioning by an automated, computer-aided machine such
as a bioprinter is advantageous. Advantages of delivery of cells or
multicellular bodies with this technology include rapid, accurate,
and reproducible placement of cells or multicellular bodies to
produce constructs exhibiting planned or pre-determined
orientations or patterns of cells, multicellular aggregates and/or
layers thereof with various compositions. Advantages also include
assured high cell density, while minimizing cell damage.
[0070] In some embodiments, the method of bioprinting is continuous
and/or substantially continuous. A non-limiting example of a
continuous bioprinting method is to dispense bio-ink (i.e., cells,
cells combined with an excipient or extrusion compound, or
aggregates of cells) from a bioprinter via a dispense tip (e.g., a
syringe, needle, capillary tube, etc.) connected to a reservoir of
bio-ink. In further non-limiting embodiments, a continuous
bioprinting method is to dispense bio-ink in a repeating pattern of
functional units. In various embodiments, a repeating functional
unit has any suitable geometry, including, for example, circles,
squares, rectangles, triangles, polygons, and irregular geometries,
thereby resulting in one or more tissue layers with planar geometry
achieved via spatial patterning of distinct bio-inks and/or void
spaces. In further embodiments, a repeating pattern of bioprinted
function units comprises a layer and a plurality of layers are
bioprinted adjacently (e.g., stacked) to form an engineered tissue
or organ with laminar geometry. In various embodiments, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more layers are bioprinted
adjacently (e.g., stacked) to form an engineered tissue or organ.
In further embodiments, one or more layers of a tissue with laminar
geometry also has planar geometry.
[0071] In some embodiments, a bioprinted functional unit repeats in
a tessellated pattern. A "tessellated pattern" is a plane of
figures that fills the plane with no overlaps and no gaps. FIG. 6A
shows an example of a functional unit that is optionally repeated
to produce the tessellation pattern depicted in FIGS. 6B-D and 7.
Advantages of continuous and/or tessellated bioprinting includes,
by way of non-limiting example, increased productivity of
bioprinted tissue. Another non-limiting, exemplary advantage is
eliminating the need to align the bioprinter with previously
deposited elements of bio-ink. In some embodiments, continuous
bioprinting facilitates printing larger tissues from a large
reservoir of bio-ink, optionally using a syringe mechanism.
Continuous bioprinting is also a convenient way to co-print
spatially-defined boundaries, using an extrusion compound, a
hydrogel, a polymer, bio-ink, or any printable material that is
capable of retaining its shape post-printing; wherein the
boundaries that are created are optionally filled in via the
bioprinting of a one or more bio-inks, thereby creating a mosaic
tissue with spatially-defined planar geometry, see for example, the
embodiment illustrated in FIG. 17.
[0072] In some embodiments, methods in continuous bioprinting
involve optimizing and/or balancing parameters such as print
height, pump speed, robot speed, or combinations thereof
independently or relative to each other. In one example, the
bioprinter head speed for deposition was 3 mm/s, with a dispense
height of 0.5 mm for the first layer and dispense height was
increased 0.4 mm for each subsequent layer. In some embodiments,
the dispense height is approximately equal to the diameter of the
bioprinter dispense tip. Without limitation a suitable and/or
optimal dispense distance does not result in material flattening or
adhering to the dispensing needle. In various embodiments, the
bioprinter dispense tip has an inner diameter of about, 20, 50,
100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,
750, 800, 850, 900, 950, 1000 .mu.m, or more, including increments
therein. In various embodiments, the bio-ink reservoir of the
bioprinter has a volume of about 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100 cubic centimeters, or more, including
increments therein. The pump speed is, in some cases, suitable
and/or optimal when the residual pressure build-up in the system is
low. Favorable pump speeds, in some cases, depend on the ratio
between the cross-sectional areas of the reservoir and dispense
needle with larger ratios requiring lower pump speeds. In some
embodiments, a suitable and/or optimal print speed enables the
deposition of a uniform line without affecting the mechanical
integrity of the material.
[0073] The inventions disclosed herein include business methods. In
some embodiments, the speed and scalability of the techniques and
methods disclosed herein are utilized to design, build, and operate
industrial and/or commercial facilities for production of
engineered tissues and/or organs for implantation or use in
generation of cell-based tools for research and development, such
as in vitro assays. In further embodiments, the engineered tissues
and/or organs and arrays thereof are produced, stored, distributed,
marketed, advertised, and sold as, for example, cellular arrays
(e.g., microarrays or chips), tissue arrays (e.g., microarrays or
chips), and kits for biological assays and high-throughput drug
screening. In other embodiments, the engineered tissues and/or
organs and arrays thereof are produced and utilized to conduct
biological assays and/or drug screening as a service.
Engineered Tissues
[0074] Disclosed herein, in some embodiments, are living,
three-dimensional tissue constructs comprising at least one
adherent cell type, wherein the at least one adherent cell type is
cohered and fused to form a tissue construct with a multi-layered
architecture. In further embodiments, at least one component of the
tissue construct was bioprinted. In some embodiments, the tissues
are vascular wall segments (see, e.g., Example 16 and FIGS. 12 and
13). Therefore, also disclosed herein, in some embodiments, are
engineered vascular wall segments comprising: smooth muscle cells;
and optionally, fibroblasts and/or endothelial cells; wherein the
cells are cohered to one another; wherein the vascular wall segment
was bioprinted and is non-tubular. In other embodiments, the
tissues are airway analogues (see, e.g., Example 15 and FIGS. 10
and 11). In some embodiments, the airway analogues comprise:
pulmonary fibroblasts and optionally, smooth muscle cells and/or
endothelial cells, wherein at least one surface of the tissue is
layered with small airway epithelial cells. In other embodiments,
the tissues are liver analogues (see, e.g., Examples 13 and 19 and
FIGS. 6A-D, 7, and 17A-B). In further embodiments, the liver tissue
analogues comprise: hepatocytes or hepatocyte-like cells and
optionally bile duct epithelial cells and optionally,
non-parenchymal cell types including, but not limited to, stellate
cells, endothelial cells, kupffer cells, immune cells, or
myofibroblasts.
[0075] Also disclosed herein, in certain embodiments, are
engineered tissues comprising cohered, mammalian cells, and further
comprising one or more layers of mammalian cells, wherein at least
one component of the tissue was bioprinted. In some embodiments,
one or more of the tissue layers is characterized by a planar
geometry, wherein multiple cell types or bio-ink types and/or void
spaces exist in spatially-defined positions in the x-y planes. In
some embodiments, the tissues are multi-layered wherein at least
one of the layers is architecturally or compositionally distinct
from the other layers, giving the tissue a characteristic laminar
geometry. In further embodiments, the layers are of similar
thickness in the z-plane. In still further embodiments, the layers
are of variable thickness in the z-plane. In further embodiments,
any single layer is one cell layer in thickness. In some
embodiments, the tissues are vascular wall segments. Therefore,
also disclosed herein, in certain embodiments, are engineered
vascular wall segments comprising cohered smooth muscle cells, and
a layer of endothelial cells on one or more surfaces, a layer of
fibroblasts on one or more surfaces, or both, wherein at least one
component of said vascular wall segment was bioprinted; and wherein
said vascular wall segment is non-tubular. In other embodiments,
the tissues are airway analogues. In some embodiments, the airway
analogues comprise: pulmonary fibroblasts and optionally, smooth
muscle cells and/or endothelial cells, wherein at least one surface
of the tissue is layered with small airway epithelial cells. In
other embodiments, the tissues are liver analogues. In further
embodiments, the liver tissue analogues comprise: hepatocytes or
hepatocyte-like cells and optionally bile duct epithelial cells and
optionally, non-parenchymal cell types including, but not limited
to, stellate cells, endothelial cells, kupffer cells, immune cells,
or myofibroblasts.
[0076] In some embodiments, the engineered tissues, including
vascular wall segments, are bioprinted, a methodology described
herein. In further embodiments, at least one component of the
engineered tissue is bioprinted. In further embodiments, the
bioprinted component comprises cohered smooth muscle cells. In
still further embodiments, additional components of the tissue are
bioprinted. In further embodiments, the additional bioprinted
layers comprise fibroblasts and/or endothelial cells. In further
embodiments, the tissues are free of any pre-formed scaffold as
described further herein at the time of manufacture or at the time
of use. In some embodiments, as a result of being fabricated by
tissue engineering techniques, including bioprinting, the tissues
of the present invention are further distinguished from tissues
developed in vivo, as part of an organism. In some embodiments, one
layer of the engineered tissue consists of interstitial tissue,
comprising various cell types such as fibroblasts, smooth muscle
cells, myofibroblasts, pericytes, and endothelial cells. In further
embodiments, the interstitial tissue is layered on one or more
surfaces with a second tissue type comprising generic or
tissue-specific endothelial or epithelial cells. In still further
embodiments, the second tissue layer is contiguous and serves as a
barrier for passage of molecules to the underlying interstitial
tissue layer.
[0077] In some embodiments, the engineered tissues, including
vascular wall segments, include any type of mammalian cell. In
various further embodiments, the tissues, including vascular wall
segments, include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20 or more cell types. In some embodiments, the
tissues include only smooth muscle cells. In some embodiments, the
tissues include smooth muscle cells and endothelial cells. Example
3 demonstrates fabrication of polytypic cylindrical bio-ink
consisting of human aortic smooth muscle cells and human aortic
endothelial cells while Example 4 demonstrates bioprinting and
fusion of such cylinders to form blood vessel wall segments (see
e.g., FIGS. 1, 2a, and 2b). Example 7 demonstrates fabrication of
polytypic cylindrical bio-ink consisting of smooth muscle cells and
endothelial cells cultured from the stromal vascular fraction of
human lipoaspirate while Example 8 demonstrates bioprinting and
fusion of such cylinders to form blood vessel wall segments. In
other embodiments, the tissues include smooth muscle cells and
fibroblasts. In yet other embodiments, the tissues include smooth
muscle cells, endothelial cells, and fibroblasts. Example 5
demonstrates fabrication of polytypic cylindrical bio-ink
consisting of human aortic smooth muscle cells, human dermal
fibroblasts, and human aortic endothelial cells while Example 6
demonstrates bioprinting and fusion of such cylinders to form blood
vessel wall segments. In some embodiments, the cells of the
engineered tissues, including vascular wall segments are "cohered"
or "adhered" to one another. In further embodiments, cohesion or
adhesion refers to cell-cell adhesion properties that bind cells,
multicellular aggregates, multicellular bodies, and/or layers
thereof.
[0078] In some embodiments, the engineered tissues, including
vascular wall segments, include one or more layers of cells on one
or more surfaces. In further embodiments, one or more layers of
cells are on one or more surfaces of the cohered smooth muscle
cells. In further various embodiments, there are 1, 2, 3, 4, 5, 6,
7, 8, 9, 10 or more layers of cells on one or more surfaces of the
cohered smooth muscle cells. In still further various embodiments,
there is at least one layer of cells on 1, 2, 3, 4 or more surfaces
of the cohered smooth muscle cells, creating a laminar geometry in
the engineered tissue. In further embodiments, one or more of the
layers is characterized by having a planar geometry. In still
further embodiments, multiple layers of the engineered tissue have
a planar geometry; wherein the planar geometries are variable among
layers or are the same. In still further embodiments, planar
geometries (x-y planes) in individual layers are aligned in the
z-plane during fabrication so that additional geometry is created
in the z-plane in the composite tissue (see, e.g., embodiments
presented in FIGS. 18D-F).
[0079] In some embodiments, a layer of tissue comprises a monolayer
of cells. In further embodiments, the monolayer is confluent. In
other embodiments, the monolayer is not confluent. In some
embodiments, a layer of cells comprises one or more sheets of
cells. In various embodiments, a sheet of cells is about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more
cells thick, including increments therein. In other various
embodiments, a sheet of cells is about 3, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,
175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,
500 or more .mu.m thick, including increments therein. In some
embodiments, a layer of tissue comprises fused aggregates of cells.
In further embodiments, prior to fusion, the aggregates of cells
have, by way of non-limiting examples, a defined shape and/or
architecture, being substantially spherical, elongate,
substantially cylindrical and ribbon-like shape. In various
embodiments, fused aggregates of cells form a layer about 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,
425, 450, 475, 500 or more .mu.m thick, including increments
therein.
[0080] In some embodiments, the one or more layers include any type
of mammalian cell. In various further embodiments, each layer
includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 or more cell types. In some embodiments, the engineered
tissues, including vascular wall segments, include one or more
layers of endothelial cells on one or more surfaces. Example 9
demonstrates construction of vascular wall segments by bioprinting
a layer of vascular media tissue comprising cylindrical smooth
muscle cell bio-ink, followed by application of a second layer of
endothelial cells to the top surface, achieved by bioprinting a
cell concentrate directly onto the SMC construct to generate a
laminar geometry that recapitulates the media and intima of the
blood vessel wall. Example 10 demonstrates construction of vascular
wall segments by bioprinting of cylindrical bio-ink comprising
human aortic smooth muscle cells followed by application of a layer
of endothelial cells to the top surface, achieved by deposition of
specifically positioned droplets of endothelial cells onto the SMC
construct. In some embodiments, the engineered tissues, including
vascular wall segments, include one or more layers of fibroblasts
on one or more surfaces.
[0081] In some embodiments, the engineered tissues, including
vascular wall segments, include one or more layers of endothelial
cells on one or more surfaces and one or more layers of fibroblasts
on one or more surfaces. In further embodiments, the one or more
layers of endothelial cells are on the same surfaces as the one or
more layers of fibroblasts. In other embodiments, the one or more
layers of endothelial cells are on surfaces distinct from surfaces
with one or more layers of fibroblasts. In further embodiments, one
or more of the layers within the multi-layered architecture is
characterized further by having planar geometry.
[0082] Example 11 demonstrates construction of vascular wall
segments by bioprinting cylindrical bio-ink comprising human aortic
smooth muscle cells directly onto a first layer of fibroblasts,
followed by application of a third layer comprising endothelial
cells to the top surface, thereby creating a tri-layered laminar
geometry wherein each layer is compositionally distinct and of
variable thickness and architecture (see, e.g., FIG. 12). The layer
of endothelial cells is applied by deposition of specifically
positioned droplets of endothelial cell suspension onto the
construct. The procedures of Example 11 result in a tri-layered
tissue comprising cohered smooth muscle cells, a layer of
fibroblasts on one surface of the smooth muscle cells, and a layer
of fibroblasts on an opposing surface of the smooth muscle cells.
The cells within each layer are cohered to each other, and the
cells positioned at the interface between layers are also cohered,
thereby bonding the individual layers together by cellular
interactions (see, e.g., FIGS. 4a and 4b).
[0083] The engineered tissues, including vascular wall segments, in
various embodiments, are any suitable size. In some embodiments,
the size of bioprinted tissues, including vascular wall segments,
change over time. In further embodiments, a bioprinted tissue
shrinks or contracts after bioprinting due to, for example, cell
migration, cell death, intercellular interactions, contraction, or
other forms of shrinkage. In other embodiments, a bioprinted tissue
grows or expands after bioprinting due to, for example, cell
migration, cell growth and proliferation, production of
extracellular matrix or other cell-produced components of native
tissue, cell/tissue maturation or other forms of expansion.
[0084] In some embodiments, the physical dimensions of the
engineered tissues, including vascular wall segments, are limited
by the capacity for nutrients, including oxygen, to diffuse into
the interior of the construct. In various embodiments, the
engineered tissues, including vascular wall segments, are at least
about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,
280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,
410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, or 1000 .mu.m in their smallest
dimension at the time of bioprinting. In various embodiments, the
engineered tissues, including vascular wall segments, are at least
about 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75,
3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5.0 mm in their
smallest dimension at the time of bioprinting. In further
embodiments, the engineered tissues, including vascular wall
segments, are between about 25 .mu.m and about 500 .mu.m in their
smallest dimension at the time of bioprinting. In other
embodiments, the engineered tissues, including vascular wall
segments, are less than 3 cm in the largest dimension at the time
of fabrication.
[0085] The engineered tissues, including vascular wall segments, in
various embodiments, are any suitable shape. In some embodiments,
the shape is selected to mimic a particular natural tissue or
organ. In further embodiments, the shape is selected to mimic a
particular pathology, condition, or disease state. In some
embodiments, the engineered tissues, including vascular wall
segments, have a shape that is substantially planar. In further
embodiments, planar tissues have any suitable planar geometry
including, by way of non-limiting examples, square, rectangle,
polygon, circle, oval, or irregular. In some embodiments, a planar
geometry is generated in an engineered tissue by positioning
specific cellular or bio-ink components and/or void spaces in the
x-y planes relative to each other. In some embodiments, the
engineered tissues, including vascular wall segments, have a shape
that is substantially a sheet or disk. In some embodiments, the
engineered vascular wall segments have a shape that is non-tubular,
being a vascular wall segment, patch, or sheet, rather than a
vascular tube.
[0086] In some embodiments, the engineered tissues, including
vascular wall segments, are secured to containment vessel by a
means suitable to fix the position of the tissue in space relative
to the containment vessel. In further embodiments, the engineered
tissues are affixed to a surface. In further embodiments, the
tissues are affixed to a biocompatible surface. In still further
embodiments, a plurality of tissues are associated by affixation to
a surface and spatially arranged to form an array, as described
herein. In some embodiments, engineered tissues, including vascular
wall segments, are subjected to shear force, caused by fluid flow,
on one or more sides (see, e.g., FIG. 13). In further embodiments,
application of shear force serves to facilitate the maturation and
development of a tissue and/or facilitate the migration,
differentiation, proliferation, deposition of extracellular matrix,
or transport of proteins or molecules into or out of cells within
the tissue.
Tissue Geometries
[0087] Native tissues are characterized by the presence of spatial
and compositional patterns driven by the cellular and extracellular
(i.e., void spaces, extracellular matrices, proteinaceous matter,
etc.) components of a tissue. Inherent challenges to tissue
engineering strategies that deploy synthetic scaffolding to achieve
three-dimensionality is the inability to reproduce both the
geometric and biologic attributes of native tissue. To date,
attempts to create native tissue-like laminar or planar geometry
within a scaffold structure while also enabling the incorporation
of cells at a density that mimics native tissue have been hampered
by technical limitations. Bioprinting overcomes both inherent
challenges (planar/laminar geometry and cell density) through the
spatially-defined deposition of bio-ink comprised of cells,
according to the examples illustrated in FIGS. 18A-F. In some
embodiments, planar geometries are created from multiple bio-ink
formulations, whereby two or more tissue components (i.e., stromal,
epithelial, vascular, bone, cartilage, parenchymal, cortical,
medullary, papillary, lobular, etc.) are fabricated in a manner
that positions each tissue component/cell population/bio-ink
formulation in a defined position relative to each other in the x,
y, and/or z planes according to the examples set forth in FIG.
18A-C. In some embodiments, the planar geometries are generated by
bioprinting. In some embodiments, the planar geometry recapitulates
at least one spatial element of glandular tissue, cancer tissue, a
tissue interface (bone:cartilage, for example), vascularized
tissue, pyramidal tissue, zonal tissue, or lobulated tissue. In
some embodiments, the planar geometry incorporates void spaces. In
further embodiments, the void spaces within the planar geometry
accommodate fluids that mimic at least one element of bodily
fluids, such as blood, lymph, bile, urine, secretions, and the
like. In further embodiments, the void spaces optionally contain
non-adherent cell types or bodily-fluid-derived components (e.g.,
blood cells, marrow cells, lymphatic cells, immune cells, cancer
cells, platelets, proteins, etc.). In still further embodiments,
non-adherent cell types of bodily-fluid-derived components
optionally exist as a component of non-void spaces having been
introduced into the cell-comprising components of the planar
geometry before, during, or after fabrication. In still further
embodiments, non-adherent cellular components or
bodily-fluid-derived components are recruited from void spaces into
cell-comprising spaces within the planar geometry as a result of
intercellular interactions or response to secreted factors.
[0088] In some embodiments, fluid flow or perfusion is optionally
initiated through the void spaces within a geometry. In some
embodiments, planar geometries enable the generation of
tissue-tissue or tissue-liquid interfaces, as highlighted in FIG.
18B. In further embodiments, the tissues are fabricated into
containers that are optically clear to enable real-time observation
of cells at the interface(s) created by the geometry.
[0089] In some embodiments, tissues comprise multiple layers
wherein at least one of the layers is architecturally or
compositionally distinct from other layers within the construct,
thereby creating a laminar architecture in the z-plane. Examples of
laminar architecture include barrier tissues that possess an
endothelial or epithelial barrier to an underlying interstitial
tissue as depicted by the examples shown in FIG. 18D-F. In some
embodiments, laminar tissues represent a portion of the wall of a
luminal or tubular structure (e.g., intestine, blood vessel, lymph
vessel, renal tubule, ureter, bladder, trachea, esophagus, airway,
fallopian tube, urethra, ductular structures, etc.). In other
embodiments, laminar tissues represent zones or layers of a tissue
(e.g., mucosal tissues, dermal tissues, renal tissues, cardiac
tissues, etc.) (see, e.g., FIGS. 8-11). In further embodiments, one
or more layers of a tissue incorporate vascular or microvascular
components. In still further embodiments, the incorporation of
vascular or microvascular components leads to the formation of
microvascular or pseudovascular networks within one or more
components of the engineered tissue. In some embodiments, one or
more components of the tissue with laminar geometry are bioprinted.
In some embodiments, one or more tissues with laminar geometry are
fabricated adjacent to each other, thereby creating a tissue
interface, such as a mucocutaneous junction as drawn in FIG.
18E.
[0090] In some embodiments, one or more layers of a multi-layered
engineered tissue with laminar geometry also comprise planar
geometry, according to the non-limiting examples set forth in FIG.
18F. In some embodiments, the same planar geometry is continued in
each layer, resulting in a three-dimensional tissue with continuous
architecture in the x, y, and z planes. In some embodiments, the
composition or planar geometry of one or more laminar layers is
varied, such that the resulting three-dimensional tissue possesses
a complex architecture in both the x, y and z planes according to
the non-limiting example of renal tubules illustrated in FIG.
18F.
Cells
[0091] Disclosed herein, in some embodiments, are engineered
tissues comprising one or more types of mammalian cells. Also
disclosed herein, in some embodiments, are engineered vascular wall
segments comprising smooth muscle cells; and optionally,
fibroblasts and/or endothelial cells. In other embodiments, the
tissues are airway analogues. In some embodiments, the airway
analogues comprise: pulmonary fibroblasts and optionally, smooth
muscle cells and/or endothelial cells, wherein at least one surface
of the tissue is layered with small airway epithelial cells. In
other embodiments, the tissues are liver analogues. In further
embodiments, the liver tissue analogues comprise: hepatocytes or
hepatocyte-like cells and optionally bile duct epithelial cells and
optionally, non-parenchymal cell types including, but not limited
to, stellate cells, endothelial cells, kupffer cells, immune cells,
or myofibroblasts.
[0092] In some embodiments, any mammalian cell is suitable for
inclusion in the engineered tissues and arrays thereof. In further
embodiments, at least one component of the engineered tissues is an
adherent cell type. In further embodiments, the mammalian cells
are, by way of non-limiting examples, contractile or muscle cells
(e.g., skeletal muscle cells, cardiomyocytes, smooth muscle cells,
and myoblasts), connective tissue cells (e.g., bone cells,
cartilage cells, fibroblasts, and cells differentiating into bone
forming cells, chondrocytes, or lymph tissues), bone marrow cells,
endothelial cells, skin cells, epithelial cells, breast cells,
vascular cells, blood cells, lymph cells, neural cells, Schwann
cells, gastrointestinal cells, liver cells, pancreatic cells, lung
cells, tracheal cells, corneal cells, genitourinary cells, kidney
cells, reproductive cells, adipose cells, parenchymal cells,
pericytes, mesothelial cells, stromal cells, undifferentiated cells
(e.g., embryonic cells, stem cells, and progenitor cells),
endoderm-derived cells, mesoderm-derived cells, ectoderm-derived
cells, cancer-derived cells and combinations thereof.
[0093] In one embodiment, the cells are smooth muscle cells. In
another embodiment, the cells are smooth muscle cells and
fibroblasts. In yet another embodiment, the cells are smooth muscle
cells and endothelial cells. In still another embodiment, the cells
are smooth muscle cells, fibroblasts, and endothelial cells. In
embodiments including more than one cell type, the cell types are
present in many suitable ratios, examples of which are described
herein.
[0094] In some embodiments, the cells are adult, differentiated
cells. In further embodiments, "differentiated cells" are cells
with a tissue-specific phenotype consistent with, for example, a
smooth muscle cell, a fibroblast, or an endothelial cell at the
time of isolation, wherein tissue-specific phenotype (or the
potential to display the phenotype) is maintained from the time of
isolation to the time of use. In other embodiments, the cells are
adult, non-differentiated cells. In further embodiments,
"non-differentiated cells" are cells that do not have, or have
lost, the definitive tissue-specific traits of for example, smooth
muscle cells, fibroblasts, or endothelial cells. In some
embodiments, non-differentiated cells include stem cells. In
further embodiments, "stem cells" are cells that exhibit potency
and self-renewal. Stem cells include, but are not limited to,
totipotent cells, pluripotent cells, multipotent cells, oligopotent
cells, unipotent cells, and progenitor cells. In various
embodiments, stem cells are embryonic stem cells, adult stem cells,
amniotic stem cells, and induced pluripotent stem cells. In yet
other embodiments, the cells are a mixture of adult, differentiated
cells and adult, non-differentiated cells.
[0095] In some embodiments, the smooth muscle cells are human
smooth muscle cells. In some embodiments, suitable smooth muscle
cells originated from tissue including, by way of non-limiting
example, blood, blood vessel, lymphatic vessel, tissue of the
digestive tract, tissue of the genitourinary tract, adipose tissue,
tissue of the respiratory tract, tissue of the reproductive system,
bone marrow, and umbilical tissue. In some embodiments, the
endothelial cells are human endothelial cells. In some embodiments,
suitable endothelial cells originate from tissue including, by way
of non-limiting example, blood, blood vessel, lymphatic vessel,
tissue of the digestive tract, tissue of the genitourinary tract,
adipose tissue, tissue of the respiratory tract, tissue of the
reproductive system, bone marrow, and umbilical tissue. In some
embodiments, the fibroblasts are human fibroblasts. In some
embodiments, suitable fibroblasts are non-vascular fibroblasts. In
other embodiments, suitable fibroblasts are derived from vascular
adventitia. In some embodiments, some or all of the cells are
derived from mammalian lipoaspirate. In further embodiments, some
or all of the cells are cultured from the stromal vascular fraction
of mammalian lipoaspirate. See Example 1.
[0096] In various embodiments, the cell types and/or source of the
cells are selected, configured, treated, or modulated based on a
specific research goal or objective. In some embodiments, one or
more specific cell types are selected, configured, treated, or
modulated to facilitate investigation of a particular disease or
condition. In some embodiments, one or more specific cell types are
selected, configured, treated, or modulated to facilitate
investigation of a disease or a condition of a particular subject.
In some embodiments, one or more specific cell types are derived
from two or more distinct human donors. In some embodiments, one or
more specific cell types are derived from a particular vertebrate
subject. In further embodiments, one or more specific cell types
are derived from a particular mammalian subject. In still further
embodiments, one or more specific cell types are derived from a
particular human subject. In further embodiments, one or more
specific cell types are derived from a particular subject with a
specific phenotype associated with disease or tissue functionality.
In still further embodiments, the subject-specific cells are
isolated from the target tissue of interest by way of biopsy or
tissue sampling. In further embodiments, the subject-specific cells
are utilized to fabricate tissue immediately after isolation. In
other embodiments, the subject-specific cells are manipulated in
vitro prior to use in the fabrication of three-dimensional tissues;
wherein the manipulation includes one or more of: expansion,
differentiation, directed differentiation, proliferation, exposure
to proteins or nucleic acids, incorporation of genetic vectors,
incorporation of genetic or non-genetic cell-tracing moieties,
de-differentiation (i.e., generation of induced pluripotent stem
cells or equivalents), cryopreservation. In some embodiments,
subject-specific cells are isolated from a tissue other than the
target tissue. In further embodiments, the subject-specific cells
require differentiation into cell types of interest within the
target tissue. In still further embodiments, subject-specific cells
that require differentiation are differentiated prior to, during,
or after fabrication into a three-dimensional structure.
Methods of Culturing Cells
[0097] The cell types used in the engineered tissues of the
invention are suitably cultured in any manner known in the art.
Methods of cell and tissue culturing are known in the art, and are
described, for example, in Freshney, R., Culture of Animal Cells: A
Manual of Basic Techniques, Wiley (1987), the contents of which are
incorporated herein by reference for such information. General
mammalian cell culture techniques, cell lines, and cell culture
systems suitably used in conjunction with the present invention are
also described in Doyle, A., Griffiths, J. B., Newell, D. G.,
(eds.) Cell and Tissue Culture: Laboratory Procedures, Wiley
(1998), the contents of which are incorporated herein by reference
for such information.
[0098] Appropriate growth conditions for mammalian cells in culture
are well known in the art. See, e.g., Example 1. Cell culture media
generally include essential nutrients and, optionally, additional
elements such as growth factors, salts, minerals, vitamins,
platelet-rich plasma, etc., that are optionally selected according
to the cell type(s) being cultured. In some embodiments, particular
ingredients are selected to enhance cell growth, differentiation,
secretion of specific proteins, etc. In general, standard growth
media include Dulbecco's Modified Eagle Medium (DMEM) or low
glucose with 110 mg/L pyruvate and glutamine, supplemented with
1-20% fetal bovine serum (FBS), calf serum, or human serum, 100
U/mL penicillin, and 0.1 mg/mL streptomycin are appropriate, as are
various other standard media well known to those in the art.
Preferably cells are cultured under sterile conditions in an
atmosphere of 1-21% O.sub.2 and preferably 3-5% CO.sub.2, at a
temperature at or near the body temperature of the animal of origin
of the cell. For example, human cells are preferably cultured at
approximately 37.degree. C.
[0099] The cells are optionally cultured with cellular
differentiation agents to induce differentiation of the cell along
the desired line. For instance, cells are optionally cultured with
growth factors, cytokines, etc. In some embodiments, the term
"growth factor" refers to a protein, a polypeptide, or a complex of
polypeptides, including cytokines, that are produced by a cell and
affect itself and/or a variety of other neighboring or distant
cells. Typically growth factors affect the growth and/or
differentiation of specific types of cells, either developmentally
or in response to a multitude of physiological or environmental
stimuli. Some, but not all, growth factors are hormones. Exemplary
growth factors are insulin, insulin-like growth factor (IGF), nerve
growth factor (NGF), vascular endothelial growth factor (VEGF),
keratinocyte growth factor (KGF), fibroblast growth factors (FGFs),
including basic FGF (bFGF), platelet-derived growth factors
(PDGFs), including PDGF-AA and PDGF-AB, hepatocyte growth factor
(HGF), transforming growth factor alpha (TGF-.alpha.), transforming
growth factor beta (TGF-.beta.), including TGF.beta.1 and
TGF.beta.3, epidermal growth factor (EGF), granulocyte-macrophage
colony-stimulating factor (GM-CSF), granulocyte colony-stimulating
factor (G-CSF), interleukin-6 (IL-6), IL-8, and the like. Growth
factors are discussed in, among other places, Molecular Cell
Biology, Scientific American Books, Darnell et al., eds., 1986;
Principles of Tissue Engineering, 2d ed., Lanza et al., eds.,
Academic Press, 2000. The skilled artisan will understand that any
and all culture-derived growth factors in the conditioned media
described herein are within the scope of the invention.
Bio-Ink and Multicellular Aggregates
[0100] Disclosed herein, in certain embodiments, are
three-dimensional living tissues, including vascular wall segments,
arrays thereof, and methods that comprise bioprinted cells. In some
embodiments, cells are bioprinted by depositing or extruding
bio-ink from a bioprinter. In some embodiments, "bio-ink" includes
liquid, semi-solid, or solid compositions comprising a plurality of
cells. In some embodiments, bio-ink comprises liquid or semi-solid
cell solutions, cell suspensions, or cell concentrations. In
further embodiments, a cell solution, suspension, or concentration
comprises a liquid or semi-solid (e.g., viscous) carrier and a
plurality of cells. In still further embodiments, the carrier is a
suitable cell nutrient media, such as those described herein. In
some embodiments, bio-ink comprises a plurality of cells that
optionally cohere into multicellular aggregates prior to
bioprinting. In further embodiments, bio-ink comprises a plurality
of cells and is bioprinted to produce a specific planar and/or
laminar geometry; wherein cohesion of the individual cells within
the bio-ink takes place before, during and/or after bioprinting. In
some embodiments, the bio-ink is produced by 1) collecting a
plurality of cells in a fixed volume; wherein the cellular
component(s) represent at least about 30% and at most 100% of the
total volume. In some embodiments, bio-ink comprises semi-solid or
solid multicellular aggregates or multicellular bodies. In further
embodiments, the bio-ink is produced by 1) mixing a plurality of
cells or cell aggregates and a biocompatible liquid or gel in a
pre-determined ratio to result in bio-ink, and 2) compacting the
bio-ink to produce the bio-ink with a desired cell density and
viscosity. In some embodiments, the compacting of the bio-ink is
achieved by centrifugation, tangential flow filtration ("TFF"), or
a combination thereof. In some embodiments, the compacting of the
bio-ink results in a composition that is extrudable, allowing
formation of multicellular aggregates or multicellular bodies. In
some embodiments, "extrudable" means able to be shaped by forcing
(e.g., under pressure) through a nozzle or orifice (e.g., one or
more holes or tubes). In some embodiments, the compacting of the
bio-ink results from growing the cells to a suitable density. The
cell density necessary for the bio-ink will vary with the cells
being used and the tissue or organ being produced. In some
embodiments, the cells of the bio-ink are cohered and/or adhered.
In some embodiments, "cohere," "cohered," and "cohesion" refer to
cell-cell adhesion properties that bind cells, multicellular
aggregates, multicellular bodies, and/or layers thereof. In further
embodiments, the terms are used interchangeably with "fuse,"
"fused," and "fusion." In some embodiments, the bio-ink
additionally comprises support material, cell culture medium (or
supplements thereof), extracellular matrix (or components thereof),
cell adhesion agents, cell death inhibitors, anti-apoptotic agents,
anti-oxidants, extrusion compounds, and combinations thereof.
[0101] In various embodiments, the cells are any suitable cell. In
further various embodiments, the cells are vertebrate cells,
mammalian cells, human cells, or combinations thereof. In some
embodiments, the type of cell used in a method disclosed herein
depends on the type of construct or tissue being produced. In some
embodiments, the bio-ink comprises one type of cell (also referred
to as a "homogeneous" or "monotypic" bio-ink). In some embodiments,
the bio-ink comprises more than one type of cell (also referred to
as a "heterogeneous" or "polytypic" bio-ink).
Cell Culture Media
[0102] In some embodiments, the bio-ink comprises a cell culture
medium. The cell culture medium is any suitable medium. In various
embodiments, suitable cell culture media include, by way of
non-limiting examples, Dulbecco's Phosphate Buffered Saline,
Earle's Balanced Salts, Hanks' Balanced Salts, Tyrode's Salts,
Alsever's Solution, Gey's Balanced Salt Solution, Kreb's-Henseleit
Buffer Modified, Kreb's-Ringer Bicarbonate Buffer, Puck's Saline,
Dulbecco's Modified Eagle's Medium, Dulbecco's Modified Eagle's
Medium/Nutrient F-12 Ham, Nutrient Mixture F-10 Ham (Ham's F-10),
Medium 199, Minimum Essential Medium Eagle, RPMI-1640 Medium, Ames'
Media, BGJb Medium (Fitton-Jackson Modification), Click's Medium,
CMRL-1066 Medium, Fischer's Medium, Glascow Minimum Essential
Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), L-15
Medium (Leibovitz), McCoy's 5A Modified Medium, NCTC Medium, Swim's
S-77 Medium, Waymouth Medium, William's Medium E, or combinations
thereof. In some embodiments, the cell culture medium is modified
or supplemented. In some embodiments, the cell culture medium
further comprises albumin, selenium, transferrins, fetuins, sugars,
amino acids, vitamins, growth factors, cytokines, hormones,
antibiotics, lipids, lipid carriers, cyclodextrins, platelet-rich
plasma, or a combination thereof.
Extracellular Matrix
[0103] In some embodiments, the bio-ink further comprises one or
more components of an extracellular matrix or derivatives thereof.
In some embodiments, "extracellular matrix" includes proteins that
are produced by cells and transported out of the cells into the
extracellular space, where they serve as a support to hold tissues
together, to provide tensile strength, and/or to facilitate cell
signaling. Examples, of extracellular matrix components include,
but are not limited to, collagens, fibronectin, laminins,
hyaluronates, elastin, and proteoglycans. For example, in some
embodiments, the multicellular aggregates contain various ECM
proteins (e.g., gelatin, fibrinogen, fibrin, collagens,
fibronectin, laminins, elastin, and/or proteoglycans). The ECM
components or derivatives of ECM components are optionally added to
the cell paste used to form the multicellular aggregate. The ECM
components or derivatives of ECM components added to the cell paste
are optionally purified from a human or animal source, or produced
by recombinant methods known in the art. Alternatively, the ECM
components or derivatives of ECM components are naturally secreted
by the cells in the elongate cellular body, or the cells used to
make the elongate cellular body are optionally genetically
manipulated by any suitable method known in the art to vary the
expression level of one or more ECM components or derivatives of
ECM components and/or one or more cell adhesion molecules or
cell-substrate adhesion molecules (e.g., selectins, integrins,
immunoglobulins, and adherins). In some embodiments, the ECM
components or derivatives of ECM components promote cohesion of the
cells in the multicellular aggregates. For example, gelatin and/or
fibrinogen is suitably added to the cell paste, which is used to
form multicellular aggregates. The fibrinogen is converted to
fibrin by the addition of thrombin.
[0104] In some embodiments, the bio-ink further comprises an agent
that encourages cell adhesion.
[0105] In some embodiments, the bio-ink further comprises an agent
that inhibits cell death (e.g., necrosis, apoptosis, or
autophagocytosis). In some embodiments, the bio-ink further
comprises an anti-apoptotic agent. Agents that inhibit cell death
include, but are not limited to, small molecules, antibodies,
peptides, peptibodies, or combination thereof. In some embodiments,
the agent that inhibits cell death is selected from: anti-TNF
agents, agents that inhibit the activity of an interleukin, agents
that inhibit the activity of an interferon, agents that inhibit the
activity of an GCSF (granulocyte colony-stimulating factor), agents
that inhibit the activity of a macrophage inflammatory protein,
agents that inhibit the activity of TGF-B (transforming growth
factor B) (see, e.g., FIGS. 15 and 16), agents that inhibit the
activity of an MMP (matrix metalloproteinase), agents that inhibit
the activity of a caspase, agents that inhibit the activity of the
MAPK/JNK signaling cascade, agents that inhibit the activity of a
Src kinase, agents that inhibit the activity of a JAK (Janus
kinase), or a combination thereof. In some embodiments, the bio-ink
comprises an anti-oxidant. In some embodiments, the bio-ink
comprises oxygen-carriers or other cell-specific nutrients.
Extrusion Compounds
[0106] In some embodiments, the bio-ink further comprises an
extrusion compound (i.e., a compound that modifies the extrusion
properties of the bio-ink). Examples of extrusion compounds
include, but are not limited to gels, hydrogels, peptide hydrogels,
amino acid-based gels, surfactant polyols (e.g., Pluronic F-127 or
PF-127), thermo-responsive polymers, hyaluronates, alginates,
extracellular matrix components (and derivatives thereof),
collagens, other biocompatible natural or synthetic polymers,
nanofibers, and self-assembling nanofibers. In some embodiments,
extrusion compounds are removed after bioprinting by physical,
chemical, or enzymatic means.
[0107] Gels, sometimes referred to as jellies, have been defined in
various ways. For example, the United States Pharmacopoeia defines
gels as semisolid systems consisting of either suspensions made up
of small inorganic particles or large organic molecules
interpenetrated by a liquid. Gels include a single-phase or a
two-phase system. A single-phase gel consists of organic
macromolecules distributed uniformly throughout a liquid in such a
manner that no apparent boundaries exist between the dispersed
macromolecules and the liquid. Some single-phase gels are prepared
from synthetic macromolecules (e.g., carbomer) or from natural gums
(e.g., tragacanth). In some embodiments, single-phase gels are
generally aqueous, but will also be made using alcohols and oils.
Two-phase gels consist of a network of small discrete
particles.
[0108] Gels, in some cases, are classified as being hydrophobic or
hydrophilic. In certain embodiments, the base of a hydrophobic gel
consists of a liquid paraffin with polyethylene or fatty oils
gelled with colloidal silica, or aluminum or zinc soaps. In
contrast, the base of hydrophobic gels usually consists of water,
glycerol, or propylene glycol gelled with a suitable gelling agent
(e.g., tragacanth, starch, cellulose derivatives, carboxyvinyl
polymers, and magnesium-aluminum silicates). In certain
embodiments, the rheology of the compositions or devices disclosed
herein is pseudo plastic, plastic, thixotropic, or dilatant.
[0109] Suitable hydrogels include those derived from collagen,
hyaluronate, fibrin, alginate, agarose, chitosan, and combinations
thereof. In other embodiments, suitable hydrogels are synthetic
polymers. In further embodiments, suitable hydrogels include those
derived from poly(acrylic acid) and derivatives thereof,
poly(ethylene oxide) and copolymers thereof, poly(vinyl alcohol),
polyphosphazene, and combinations thereof. In various specific
embodiments, the confinement material is selected from: hydrogel,
NovoGel.TM., agarose, alginate, gelatin, Matrigel.TM., hyaluronan,
poloxamer, peptide hydrogel, poly(isopropyl n-polyacrylamide),
polyethylene glycol diacrylate (PEG-DA), hydroxyethyl methacrylate,
polydimethylsiloxane, polyacrylamide, poly(lactic acid), silicon,
silk, or combinations thereof.
[0110] In some embodiments, hydrogel-based extrusion compounds are
thermoreversible gels (also known as thermo-responsive gels or
thermogels). In some embodiments, a suitable thermoreversible
hydrogel is not a liquid at room temperature. In specific
embodiments, the gelation temperature (Tgel) of a suitable hydrogel
is about 10.degree. C., 11.degree. C., 12.degree. C., 13.degree.
C., 14.degree. C., 15.degree. C., 16.degree. C., 17.degree. C.,
18.degree. C., 19.degree. C., 20.degree. C., 21.degree. C.,
22.degree. C., 23.degree. C., 24.degree. C., 25.degree. C.,
26.degree. C., 27.degree. C., 28.degree. C., 29.degree. C.,
30.degree. C., 31.degree. C., 32.degree. C., 33.degree. C.,
34.degree. C., 35.degree. C., 36.degree. C., 37.degree. C.,
38.degree. C., 39.degree. C., 40.degree. C., including increments
therein. In certain embodiments, the Tgel of a suitable hydrogel is
about 10.degree. C. to about 40.degree. C. In further embodiments,
the Tgel of a suitable hydrogel is about 20.degree. C. to about
30.degree. C. In some embodiments, the bio-ink (e.g., comprising
hydrogel, one or more cell types, and other additives, etc.)
described herein is not a liquid at room temperature. In some
embodiments, a suitable thermoreversible hydrogel is not a liquid
at mammalian body temperature. In specific embodiments, the
gelation temperature (Tgel) of a suitable hydrogel is about
22.degree. C., 23.degree. C., 24.degree. C., 25.degree. C.,
26.degree. C., 27.degree. C., 28.degree. C., 29.degree. C.,
30.degree. C., 31.degree. C., 32.degree. C., 33.degree. C.,
34.degree. C., 35.degree. C., 36.degree. C., 37.degree. C.,
38.degree. C., 39.degree. C., 40.degree. C., 41.degree. C.,
41.degree. C., 43.degree. C., 44.degree. C., 45.degree. C.,
46.degree. C., 47.degree. C., 48.degree. C., 49.degree. C.,
50.degree. C., 51.degree. C., 52.degree. C., including increments
therein. In certain embodiments, the Tgel of a suitable hydrogel is
about 22.degree. C. to about 52.degree. C. In further embodiments,
the Tgel of a suitable hydrogel is about 32.degree. C. to about
42.degree. C. In some embodiments, the bio-ink (e.g., comprising
hydrogel, one or more cell types, and other additives, etc.)
described herein is not a liquid at mammalian body temperature. In
specific embodiments, the gelation temperature (Tgel) of a bio-ink
described herein is about 10.degree. C., about 15.degree. C., about
20.degree. C., about 25.degree. C., about 30.degree. C., about
35.degree. C., about 40.degree. C., about 45.degree. C., about
50.degree. C., about 55.degree. C., including increments therein.
In a specific embodiment, the Tgel of a bio-ink described herein is
about 10.degree. C. to about 15.degree. C. In another specific
embodiment, the Tgel of a bio-ink described herein is about
15.degree. C. to about 20.degree. C. In another specific
embodiment, the Tgel of a bio-ink described herein is about
20.degree. C. to about 25.degree. C. In another specific
embodiment, the Tgel of a bio-ink described herein is about
25.degree. C. to about 30.degree. C. In another specific
embodiment, the Tgel of a bio-ink described herein is about
30.degree. C. to about 35.degree. C. In another specific
embodiment, the Tgel of a bio-ink described herein is about
35.degree. C. to about 40.degree. C. In another specific
embodiment, the Tgel of a bio-ink described herein is about
40.degree. C. to about 45.degree. C. In another specific
embodiment, the Tgel of a bio-ink described herein is about
45.degree. C. to about 50.degree. C.
[0111] Polymers composed of polyoxypropylene and polyoxyethylene
form thermoreversible gels when incorporated into aqueous
solutions. These polymers have the ability to change from the
liquid state to the gel state at temperatures maintainable in a
bioprinter apparatus. The liquid state-to-gel state phase
transition is dependent on the polymer concentration and the
ingredients in the solution.
[0112] Poloxamer 407 (Pluronic F-127 or PF-127) is a nonionic
surfactant composed of polyoxyethylene-polyoxypropylene copolymers.
Other poloxamers include 188 (F-68 grade), 237 (F-87 grade), 338
(F-108 grade). Aqueous solutions of poloxamers are stable in the
presence of acids, alkalis, and metal ions. PF-127 is a
commercially available polyoxyethylene-polyoxypropylene triblock
copolymer of general formula E106 P70 E106, with an average molar
mass of 13,000. The polymer is optionally further purified by
suitable methods that will enhance gelation properties of the
polymer. It contains approximately 70% ethylene oxide, which
accounts for its hydrophilicity. It is one of the series of
poloxamer ABA block copolymers. PF-127 has good solubilizing
capacity, low toxicity and is, therefore, considered a suitable
extrusion compound.
[0113] In some embodiments, the viscosity of the hydrogels and
bio-inks presented herein is measured by any means described. For
example, in some embodiments, an LVDV-II+CP Cone Plate Viscometer
and a Cone Spindle CPE-40 is used to calculate the viscosity of the
hydrogels and bio-inks. In other embodiments, a Brookfield (spindle
and cup) viscometer is used to calculate the viscosity of the
hydrogels and bio-inks. In some embodiments, the viscosity ranges
referred to herein are measured at room temperature. In other
embodiments, the viscosity ranges referred to herein are measured
at body temperature (e.g., at the average body temperature of a
healthy human).
[0114] In further embodiments, the hydrogels and/or bio-inks are
characterized by having a viscosity of between about 500 and
1,000,000 centipoise, between about 750 and 1,000,000 centipoise;
between about 1000 and 1,000,000 centipoise; between about 1000 and
400,000 centipoise; between about 2000 and 100,000 centipoise;
between about 3000 and 50,000 centipoise; between about 4000 and
25,000 centipoise; between about 5000 and 20,000 centipoise; or
between about 6000 and 15,000 centipoise.
[0115] In some embodiments, the bio-ink comprises cells and
extrusion compounds suitable for continuous bioprinting. In
specific embodiments, the bio-ink has a viscosity of about 1500
mPas. In some embodiments, a mixture of Pluronic F-127 and cellular
material is suitable for continuous bioprinting. Such a bio-ink is
suitably prepared by dissolving Pluronic F-127 powder by continuous
mixing in cold (4.degree. C.) phosphate buffered saline (PBS) over
48 hours to 30% (w/v). Pluronic F-127 is also suitably dissolved in
water. In some embodiments, cells are cultivated and expanded using
standard sterile cell culture techniques. In further embodiments,
the cells are pelleted at 200 g for example, and re-suspended in
the 30% Pluronic F-127 and aspirated into a reservoir affixed to a
bioprinter where it is, in some embodiments, allowed to solidify at
a gelation temperature from about 10 to about 25.degree. C.
Gelation of the bio-ink prior to bioprinting is optional. The
bio-ink, including bio-ink comprising Pluronic F-127 is optionally
dispensed as a liquid.
[0116] In various embodiments, the concentration of Pluronic F-127
is any value with suitable viscosity and/or cytotoxicity
properties. In some embodiments, a suitable concentration of
Pluronic F-127 is able to support weight while retaining its shape
when bioprinted. In some embodiments, the concentration of Pluronic
F-127 is about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, or about 50%. In some embodiments,
the concentration of Pluronic F-127 is between about 30% and about
40%, or between about 30% and about 35%.
[0117] In some embodiments, the non-cellular components of the
bio-ink (e.g., extrusion compounds, etc.) are removed prior to use.
In further embodiments, the non-cellular components are, for
example, hydrogels, peptide hydrogels, amino acid-based gels,
surfactant polyols, thermo-responsive polymers, hyaluronates,
alginates, collagens, or other biocompatible natural or synthetic
polymers. In still further embodiments, the non-cellular components
are removed by physical, chemical, or enzymatic means. In some
embodiments, a proportion of the non-cellular components remain
associated with the cellular components at the time of use.
[0118] In some embodiments, the cells are pre-treated to increase
cellular interaction. For example, cells are suitably incubated
inside a centrifuge tube after centrifugation in order to enhance
cell-cell interactions prior to shaping the bio-ink.
Exemplary Cell Ratios
[0119] In some embodiments, the bio-ink comprises multicellular
bodies, which further comprise smooth muscle cells and endothelial
cells. In further embodiments, the ratio of smooth muscle cells to
endothelial cells is any suitable ratio. In still further
embodiments, the ratio of smooth muscle cells to endothelial cells
is about 90:10 to about 60:40. In a particular embodiment, the
multicellular bodies comprise smooth muscle cells and endothelial
cells and the ratio of smooth muscle cells to endothelial cells is
about 85:15. In another particular embodiment, the multicellular
bodies comprise smooth muscle cells and endothelial cells and the
ratio of smooth muscle cells to endothelial cells is about
70:30.
[0120] In some embodiments, the bio-ink comprises multicellular
bodies, which further comprise smooth muscle cells and fibroblasts.
In further embodiments, the ratio of smooth muscle cells to
fibroblasts is any suitable ratio. In still further embodiments,
the ratio of smooth muscle cells to fibroblasts is about 90:10 to
about 60:40.
[0121] In some embodiments, the bio-ink comprises multicellular
bodies, which further comprise smooth muscle cells, fibroblasts,
and endothelial cells. In further embodiments, the ratio of smooth
muscle cells, fibroblasts, and endothelial cells is any suitable
ratio. In still further embodiments, the ratio of smooth muscle
cells to fibroblasts and endothelial cells is about 70:25:5.
Self-Sorting of Cells
[0122] In some embodiments, multicellular aggregates used to form
the construct or tissue comprises all cell types to be included in
the engineered tissue (e.g., endothelial cells, smooth muscle
cells, fibroblasts, etc.); in such an example each cell type
migrates to an appropriate position (e.g., during maturation) to
form the engineered tissue, such as a vascular wall segment. In
other embodiments, the multicellular aggregates used to form the
structure comprises fewer than all the cell types to be included in
the engineered tissue. In some embodiments, cells of each type are
uniformly distributed within a multicellular aggregates, or region
or layer of the tissue. In other embodiments, cells of each type
localize to particular regions within a multicellular aggregate or
layers or regions of the tissue.
[0123] For example, in the case of an engineered vascular wall
segment (e.g., vascular tissue sheet) comprising smooth muscle
cells and endothelial cells in a suitable ratio (e.g., 85:15,
70:30, etc.), neighboring, bioprinted cohered polytypic cylindrical
bio-ink units fuse. During maturation, endothelial cells localize
to some extent to the periphery of the construct and collagen is
formed. See, e.g., FIGS. 1, 3, and 4a. By way of further example,
in the case of a bioprinted vascular wall segment comprising smooth
muscle cells, fibroblasts, and endothelial cells in a suitable
ratio (e.g., 70:25:5, etc.), bioprinted polytypic cylindrical
bio-ink fuse and endothelial cells localize to some extent to the
periphery of the construct. In some embodiments, localization of
cell types within a construct mimics the layered structure of in
vivo or ex vivo mammalian tissues. In further embodiments, for
example in an engineered vascular wall segment, localization of
cell types within a construct forms putative tunica intima, tunica
media, and tunica adventitia.
[0124] In some embodiments, the sorting or self-sorting of cells is
accelerated, enhanced, or augmented by the application of one or
more layers of cells. For example, in some embodiments, a construct
bioprinted with multicellular aggregates including smooth muscle
cells and endothelial cells is further subjected to application of
a layer of endothelial cells on one or more surfaces of the
construct. In further embodiments, the result is augmentation of
the layering produced by the localization of the endothelial cells
to the periphery of the construct.
Pre-Formed Scaffold
[0125] In some embodiments, disclosed herein are engineered,
implantable tissues and organs that are free or substantially free
of any pre-formed scaffold. In further embodiments, "scaffold"
refers to synthetic scaffolds such as polymer scaffolds and porous
hydrogels, non-synthetic scaffolds such as pre-formed extracellular
matrix layers, dead cell layers, and decellularized tissues, and
any other type of pre-formed scaffold that is integral to the
physical structure of the engineered tissue and/or organ and not
removed from the tissue and/or organ. In still further embodiments,
decellularized tissue scaffolds include decellularized native
tissues or decellularized cellular material generated by cultured
cells in any manner; for example, cell layers that are allowed to
die or are decellularized, leaving behind the ECM they produced
while living.
[0126] In some embodiments, the engineered tissues, including
vascular wall segments, and arrays thereof do not utilize any
pre-formed scaffold, e.g., for the formation of the tissue, any
layer of the tissue, or formation of the tissue's shape. As a
non-limiting example, the engineered tissues of the present
invention do not utilize any pre-formed, synthetic scaffolds such
as polymer scaffolds, pre-formed extracellular matrix layers, or
any other type of pre-formed scaffold at the time of manufacture or
at the time of use. In some embodiments, the engineered tissues are
substantially free of any pre-formed scaffolds. In further
embodiments, the cellular components of the tissues contain a
detectable, but trace or trivial amount of scaffold, e.g., less
than 2.0%, less than 1.0%, or less than 0.5% of the total
composition. In still further embodiments, trace or trivial amounts
of scaffold are insufficient to affect long-term behavior of the
tissue, or array thereof, or interfere with its primary biological
function. In additional embodiments, scaffold components are
removed post-printing, by physical, chemical, or enzymatic methods,
yielding an engineered tissue that is free or substantially-free of
scaffold components.
[0127] In some embodiments, the engineered tissues free, or
substantially free, of pre-formed scaffold disclosed herein are in
stark contrast to those developed with certain other methods of
tissue engineering in which a scaffolding material is first formed,
and then cells are seeded onto the scaffold, and subsequently the
cells proliferate to fill and take the shape of the scaffold for
example. In one aspect, the methods of bioprinting described herein
allow production of viable and useful tissues that are free or
substantially free of pre-formed scaffold. In another aspect, the
cells of the invention are, in some embodiments, held in a desired
three-dimensional shape using a confinement material. The
confinement material is distinct from a scaffold at least in the
fact that the confinement material is temporary and/or removable
from the cells and/or tissue.
Arrays
[0128] In some embodiments, disclosed herein are arrays of
engineered tissues, including vascular wall segments. In some
embodiments, an "array" is a scientific tool including an
association of multiple elements spatially arranged to allow a
plurality of tests to be performed on a sample, one or more tests
to be performed on a plurality of samples, or both. In some
embodiments, the arrays are adapted for, or compatible with,
screening methods and devices, including those associated with
medium- or high-throughput screening. In further embodiments, an
array allows a plurality of tests to be performed simultaneously.
In further embodiments, an array allows a plurality of samples to
be tested simultaneously. In some embodiments, the arrays are
cellular microarrays. In further embodiments, a cellular microarray
is a laboratory tool that allows for the multiplex interrogation of
living cells on the surface of a solid support. In other
embodiments, the arrays are tissue microarrays. In further
embodiments, tissue microarrays include a plurality of separate
tissues or tissue samples assembled in an array to allow the
performance of multiple biochemical, metabolic, molecular, or
histological analyses.
[0129] In some embodiments, the engineered tissues, including
vascular wall segments each exist in a well of a biocompatible
multi-well container (see, e.g., FIG. 14). In some embodiments,
each tissue is placed into a well. In other embodiments, each
tissue is bioprinted into a well. In further embodiments, the wells
are coated. In various further embodiments, the wells are coated
with one or more of: a biocompatible hydrogel, one or more
proteins, one or more chemicals, one or more peptides, one or more
antibodies, and one or more growth factors, including combinations
thereof. In some embodiments, the wells are coated with
NovoGel.TM.. In other embodiments, the wells are coated with
agarose. In some embodiments, each tissue exists on a porous,
biocompatible membrane within a well of a biocompatible multi-well
container. In some embodiments, each well of a multi-well container
contains two or more tissues.
[0130] In some embodiments, the engineered tissues, including
vascular wall segments are secured to a biocompatible surface on
one or more sides. Many methods are suitable to secure a tissue to
a biocompatible surface. In various embodiments, a tissue is
suitably secured to a biocompatible surface, for example, along one
or more entire sides, only at the edges of one or more sides, or
only at the center of one or more sides. In various further
embodiments, a tissue is suitably secured to a biocompatible
surface with a holder or carrier integrated into the surface or
associated with the surface. In various further embodiments, a
tissue is suitably secured to a biocompatible surface with one or
more pinch-clamps or plastic nubs integrated into the surface or
associated with the surface. In some embodiments, a tissue is
suitably secured to a biocompatible surface by cell-attachment to a
porous membrane. In some embodiments, the engineered tissues,
including vascular wall segments are held in an array configuration
by affixation to a biocompatible surface on one or more sides. In
further embodiments, the tissue is affixed to a biocompatible
surface on 1, 2, 3, 4, or more sides. In some embodiments, the
biocompatible surface any surface that does not pose a significant
risk of injury or toxicity to the tissue or an organism contacting
the tissue. In further embodiments, the biocompatible surface is
any surface suitable for traditional tissue culture methods.
Suitable biocompatible surfaces include, by way of non-limiting
examples, treated plastics, membranes, porous membranes, coated
membranes, coated plastics, metals, coated metals, glass, treated
glass, and coated glass, wherein suitable coatings include
hydrogels, ECM components, chemicals, proteins, etc., and coatings
or treatments provide a means to stimulate or prevent cell and
tissue adhesion to the biocompatible surface.
[0131] In some embodiments, securing of an engineered tissue to a
biocompatible surface on one or more sides facilitates subjecting
the tissue to shear force, caused by fluid flow. In further
embodiments, the engineered tissues, including vascular wall
segments, are subjected to shear force, caused by fluid flow. In
various embodiments, the engineered tissues are subjected to shear
force on 1, 2, 3, 4, or more sides (see, e.g., FIG. 13).
[0132] In some embodiments, the arrays of engineered tissues,
including vascular wall segments, comprise an association of two or
more elements. In various embodiments, the arrays comprise an
association of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,
400, 425, 450, 475, or 500 elements, including increments therein.
In further embodiments, each element comprises one or more cells,
multicellular aggregates, tissues, organs, or combinations
thereof.
[0133] In some embodiments, the arrays of engineered tissues,
including vascular wall segments, comprise multiple elements
spatially arranged in a pre-determined pattern. In further
embodiments, the pattern is any suitable spatial arrangement of
elements. In various embodiments, patterns of arrangement include,
by way of non-limiting examples, a two-dimensional grid, a
three-dimensional grid, one or more lines, arcs, or circles, a
series of rows or columns, and the like. In further embodiments,
the pattern is chosen for compatibility with high-throughput
biological assay or screening methods or devices.
[0134] In various embodiments, the cell types and/or source of the
cells used to fabricate one or more tissues in an array are
selected based on a specific research goal or objective. In further
various embodiments, the specific tissues in an array are selected
based on a specific research goal or objective. In some
embodiments, one or more specific engineered tissues are included
in an array to facilitate investigation of a particular disease or
condition. In some embodiments, one or more specific engineered
tissues are included in an array to facilitate investigation of a
disease or a condition of a particular subject. In further
embodiments, one or more specific engineered tissues within the
array are generated with one or more cell types derived from two or
more distinct human donors. In some embodiments, each tissue within
the array is substantially similar with regard to cell types,
sources of cells, layers of cells, ratios of cells, methods of
construction, size, shape, and the like. In other embodiments, one
or more of the tissues within the array is unique with regard to
cell types, sources of cells, layers of cells, ratios of cells,
methods of construction, size, shape, and the like. In various
embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175,
200, 225, 250, 275, 300, or more of the tissues within the array,
including increments therein, is/are unique. In other various
embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% of the tissues within the array, including
increments therein, is/are unique.
[0135] In some embodiments, one or more tissues within an array
represent one or more specific tissues in the human body. In
further embodiments, one or more individual tissues within an array
represent human tissues including, by way of non-limiting example,
blood or lymph vessel, muscle, uterus, nerve, mucous membrane,
mesothelium, omentum, cornea, skin, liver, kidney, heart, trachea,
lung, bone, bone marrow, adipose, connective tissue, bladder,
breast, pancreas, spleen, brain, esophagus, stomach, intestine,
colon, rectum, ovary, prostate, tumor, endoderm, ectoderm, and
mesoderm. In one embodiment, the tissues within an array are
selected to represent all the major tissue types in a subject.
[0136] In some embodiments, each tissue within the array is
maintained independently in culture. In further embodiments, the
culture conditions of each tissue within the array are such that
they are isolated from the other tissues and cannot exchange media
or factors soluble in the media. In other embodiments, two or more
individual tissues within the array exchange soluble factors. In
further embodiments, the culture conditions of two or more
individual tissues within the array are such that they exchange
media and factors soluble in the media with other tissues. In
various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,
175, 200, 225, 250, 275, 300, or more of the tissues within the
array, including increments therein, exchange media and/or soluble
factors. In other various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the tissues within
the array, including increments therein, exchange media and/or
soluble factors.
In Vitro Assays
[0137] In some embodiments, the engineered tissues, including
vascular wall segments, and arrays disclosed herein are for use in
in vitro assays. In some embodiments, an "assay" is a procedure for
testing or measuring the presence or activity of a substance (e.g.,
a chemical, molecule, biochemical, drug, etc.) in an organic or
biologic sample (e.g., cell aggregate, tissue, organ, organism,
etc.). In further embodiments, assays include qualitative assays
and quantitative assays. In still further embodiments, a
quantitative assay measures the amount of a substance in a
sample.
[0138] In various embodiments, the engineered tissues, including
vascular wall segments and arrays are for use in, by way of
non-limiting examples, image-based assays, measurement of secreted
proteins, expression of markers, and production of proteins. In
various further embodiments, the engineered tissues, including
vascular wall segments, and arrays are for use in assays to detect
or measure one or more of: molecular binding (including radioligand
binding), molecular uptake, activity (e.g., enzymatic activity and
receptor activity, etc.), gene expression, protein expression,
receptor agonism, receptor antagonism, cell signaling, apoptosis,
chemosensitivity, transfection, cell migration, chemotaxis, cell
viability, cell proliferation, safety, efficacy, metabolism,
toxicity, and abuse liability.
[0139] In some embodiments, the engineered tissues, including
vascular wall segments, and arrays are for use in immunoassays. In
further embodiments, immunoassays are competitive immunoassays or
noncompetitive immunoassays. In a competitive immunoassay, for
example, the antigen in a sample competes with labeled antigen to
bind with antibodies and the amount of labeled antigen bound to the
antibody site is then measured. In a noncompetitive immunoassay
(also referred to as a "sandwich assay"), for example, antigen in a
sample is bound to an antibody site; subsequently, labeled antibody
is bound to the antigen and the amount of labeled antibody on the
site is then measured.
[0140] In some embodiments, the engineered tissues, including
vascular wall segments, and arrays are for use in enzyme-linked
immunosorbent assays (ELISA). In further embodiments, an ELISA is a
biochemical technique used to detect the presence of an antibody or
an antigen in a sample. In ELISA, for example, at least one
antibody with specificity for a particular antigen is utilized. By
way of further example, a sample with an unknown amount of antigen
is immobilized on a solid support (e.g., a polystyrene microtiter
plate) either non-specifically (via adsorption to the surface) or
specifically (via capture by another antibody specific to the same
antigen, in a "sandwich" ELISA). By way of still further example,
after the antigen is immobilized, the detection antibody is added,
forming a complex with the antigen. The detection antibody is, for
example, covalently linked to an enzyme, or is itself detected by a
secondary antibody that is linked to an enzyme through
bioconjugation.
[0141] For example, in some embodiments, an array, microarray, or
chip of cells, multicellular aggregates, or tissues is used for
drug screening or drug discovery. In further embodiments, an array,
microarray, or chip of tissues is used as part of a kit for drug
screening or drug discovery. In some embodiments, each vascular
wall segment exists within a well of a biocompatible multi-well
container, wherein the container is compatible with one or more
automated drug screening procedures and/or devices. In further
embodiments, automated drug screening procedures and/or devices
include any suitable procedure or device that is computer or
robot-assisted.
[0142] In further embodiments, arrays for drug screening assays or
drug discovery assays are used to research or develop drugs
potentially useful in any therapeutic area. In still further
embodiments, suitable therapeutic areas include, by way of
non-limiting examples, infectious disease, hematology, oncology,
pediatrics, cardiology, central nervous system disease, neurology,
gastroenterology, hepatology, urology, infertility, ophthalmology,
nephrology, orthopedics, pain control, psychiatry, pulmonology,
vaccines, wound healing, physiology, pharmacology, dermatology,
gene therapy, toxicology, and immunology.
Methods
[0143] Disclosed herein, in some embodiments, are methods for
constructing a living, three-dimensional tissue construct
comprising the steps of bioprinting bio-ink comprising at least one
adherent cell type into or onto a form, and fusing of the bio-ink
into a living, three-dimensional tissue construct. In further
embodiments, the tissue construct is for in vitro use. In still
further embodiments, the tissue construct is not a vascular
tube.
[0144] Also disclosed herein, in some embodiments, are methods of
constructing tissues, including vascular wall segments, comprising
the steps of: preparing cohered multicellular aggregates comprising
smooth muscle cells; placing said cohered multicellular aggregates
onto a support; and incubating said multicellular aggregates to
allow them to cohere and form a tissue such as a vascular wall
segment; wherein said incubation has a duration of about 2 hours to
about 10 days. In some embodiments, the methods utilize
bioprinting. In further embodiments, the methods produce engineered
tissues, including vascular wall segments, free or substantially
free of any pre-formed scaffold.
[0145] Also disclosed herein, in some embodiments, are methods of
constructing living, three-dimensional tissues, including vascular
wall segments, comprising the steps of: preparing one or more
cohered multicellular aggregates comprising mammalian cells;
placing said one or more cohered multicellular aggregates onto a
support; applying, to said one or more cohered multicellular
aggregates, one or more of: a layer of a first type of mammalian
cells on one or more external surfaces; a layer of a second type of
mammalian cells on one or more external surfaces; and incubating
said one or more multicellular aggregates to allow them to cohere
and to form a tissue; wherein said incubation has a duration of
about 2 hours to about 10 days. In some embodiments, the methods
utilize bioprinting. In further embodiments, the methods produce
engineered tissues, including vascular wall segments, free or
substantially free of any pre-formed scaffold.
[0146] Also disclosed herein, in some embodiments, are methods of
constructing living, three-dimensional tissue constructs comprising
the steps of: preparing one or more cohered multicellular
aggregates comprising mammalian cells; placing said one or more
cohered multicellular aggregates onto a support to form at least
one of: at least one layer comprising a plurality of cell types,
the cell types spatially arranged relative to each other to create
a planar geometry; and a plurality of layers, at least one layer
compositionally or architecturally distinct from at least one other
layer to create a laminar geometry; and incubating said one or more
multicellular aggregates to allow them to cohere and to form a
living, three-dimensional tissue construct.
Preparing Cohered Multicellular Aggregates
[0147] In some embodiments, the methods involve preparing cohered
multicellular aggregates comprising one or more types of mammalian
cells. In some embodiments, the methods involve preparing cohered
multicellular aggregates comprising smooth muscle cells. In some
embodiments, the methods involve preparing cohered multicellular
aggregates further comprising endothelial cells. See, e.g.,
Examples 3, 4, and 7. In some embodiments, the methods involve
preparing cohered multicellular aggregates further comprising
fibroblasts. See, e.g., Examples 5 and 6.
[0148] There are various ways to make multicellular aggregates
having the characteristics described herein. In some embodiments, a
multicellular aggregate is fabricated from a cell paste containing
a plurality of living cells or with a desired cell density and
viscosity. In further embodiments, the cell paste is shaped into a
desired shape and a multicellular body formed through maturation
(e.g., incubation). In some embodiments, the multicellular
aggregates are substantially cylindrical. In some embodiments, the
multicellular aggregates are substantially ribbon-shaped. In some
embodiments, the multicellular aggregates are substantially
spherical. In other embodiments, the engineered tissues are
constructed from multicellular aggregates with a range of shapes.
In a particular embodiment, an elongate multicellular body is
produced by shaping a cell paste including a plurality of living
cells into an elongate shape (e.g., a cylinder, a ribbon, etc.). In
further embodiments, the cell paste is incubated in a controlled
environment to allow the cells to adhere and/or cohere to one
another to form the elongate multicellular body. In another
particular embodiment, a multicellular body is produced by shaping
a cell paste including a plurality of living cells in a device that
holds the cell paste in a three-dimensional shape. In further
embodiments, the cell paste is incubated in a controlled
environment while it is held in the three dimensional shape for a
sufficient time to produce a body that has sufficient cohesion to
support itself on a flat surface.
[0149] In various embodiments, a cell paste is provided by: 1)
collecting cells or cell aggregates (of one or more cell types) and
a biocompatible gel or liquid, such as cell culture medium (e.g.,
in a pre-determined ratio) to result in a cell suspension, and 2)
compacting the cellular suspension to produce a cell paste with a
desired cell density and viscosity. In various embodiments,
compacting is achieved by a number of methods, such as by
concentrating a particular cell suspension that resulted from cell
culture to achieve the desired cell concentration (density),
viscosity, and consistency required for the cell paste. In a
particular embodiment, a relatively dilute cell suspension from
cell culture is centrifuged for a determined time to achieve a cell
concentration in the pellet that allows shaping in a mold.
Tangential flow filtration ("TFF") is another suitable method of
concentrating or compacting the cells. In some embodiments,
compounds are combined with the cell suspension to lend the
extrusion properties required. Suitable compounds include, by way
of non-limiting examples, surfactant polyols, collagens, hydrogels,
peptide hydrogels, amino acid-based gels, Matrigel.TM., nanofibers,
self-assembling nanofibers, gelatin, fibrinogen, etc.
[0150] In some embodiments, the cell paste is produced by mixing a
plurality of living cells with a tissue culture medium, and
compacting the living cells (e.g., by centrifugation). One or more
ECM components (or derivative of an ECM component) is optionally
included by, resuspending the cell pellet in one or more
physiologically acceptable buffers containing the ECM component(s)
(or derivative(s) of ECM component(s)) and the resulting cell
suspension centrifuged again to form a cell paste.
[0151] In some embodiments, the cell density of the cell paste
desired for further processing varies with cell types. In further
embodiments, interactions between cells determine the properties of
the cell paste, and different cell types will have a different
relationship between cell density and cell-cell interaction. In
still further embodiments, the cells are pre-treated to increase
cellular interactions before shaping the cell paste. For example,
in some cases, cells are incubated inside a centrifuge tube after
centrifugation in order to enhance cell-cell interactions prior to
shaping the cell paste. In some embodiments, the cell paste is
shaped concomitantly with bioprinting; wherein the cohesion of
individual cells to each other to form bio-ink occurs during or
after bioprinting.
[0152] In various embodiments, many methods are used to shape the
cell paste. For example, in a particular embodiment, the cell paste
is manually molded or pressed (e.g., after
concentration/compaction) to achieve a desired shape. By way of a
further example, the cell paste is taken up (e.g., aspirated) into
an instrument, such as a micropipette (e.g., a capillary pipette),
that shapes the cell paste to conform to an interior surface of the
instrument. The cross-sectional shape of the micropipette (e.g.,
capillary pipette) is alternatively circular, square, rectangular,
triangular, or other non-circular cross-sectional shape. In some
embodiments, the cell paste is shaped by depositing it into a
preformed mold, such as a plastic mold, metal mold, or a gel mold.
In some embodiments, centrifugal casting or continuous casting is
used to shape the cell paste. In some embodiments, the shaping of
the bio-ink occurs concomitantly or after bioprinting. In further
embodiments, the shaping of the bio-ink occurs as the result of a
co-printed mold; wherein the mold is optionally deposited via
bioprinting; wherein the mold comprises one or more of: gel,
hydrogel, synthetic polymer, carbohydrate, protein, or mammalian
cells. In still further embodiments, one or more components of the
co-printed mold are removed after bioprinting; wherein the removal
method is selected from one of: physical means, solubilization with
aqueous media; chemical treatment; enzymatic treatment; modulating
temperature.
[0153] In some embodiments, multicellular aggregates of a defined
shape are also suitable to build the tissues, including vascular
wall segments, described herein. Spherical multicellular aggregates
are optionally generated by a variety of methods, including, but
not limited to, cellular self-assembly, the use of molds, and
hanging drop methods. In further embodiments, a method to produce
substantially spherical multicellular aggregates comprises the
steps of 1) providing a cell paste containing a plurality of
pre-selected cells or cell aggregates with a desired cell density
and viscosity, 2) manipulating the cell paste into a cylindrical
shape, 3) cutting cylinders into equal fragments, 4) optionally
letting the fragments round up overnight on a gyratory shaker, and
5) forming the substantially spherical multicellular aggregates
through maturation. In further embodiments, cellular aggregates are
generated via acoustic focusing methodologies.
[0154] In some embodiments, a partially adhered and/or cohered cell
paste is used for bioprinting; wherein cohesion and bio-ink
formation occurs primarily post-printing. In other embodiments, the
cellular paste is shaped in a first step prior to bioprinting. In
further embodiments, the cell paste is transferred from the first
shaping device (e.g., capillary pipette) to a second shaping device
(e.g., a mold) that allows nutrients and/or oxygen to be supplied
to the cells while they are retained in the second shaping device
for an additional maturation period. One example of a suitable
shaping device that allows the cells to be supplied with nutrients
and oxygen is a mold for producing a plurality of multicellular
aggregates (e.g., substantially identical multicellular
aggregates). By way of further example, such a mold includes a
biocompatible substrate made of a material that is resistant to
migration and ingrowth of cells into the substrate and resistant to
adherence of cells to the substrate. In various embodiments, the
substrate is suitably be made of Teflon.RTM. (PTFE), stainless
steel, NovoGel.TM., agarose, polyethylene glycol, glass, metal,
plastic, or gel materials (e.g., agarose or other hydrogels), and
similar materials. In some embodiments, the mold is also suitably
configured to allow supplying tissue culture media to the cell
paste (e.g., by dispensing tissue culture media onto the top of the
mold).
[0155] Thus, in embodiments where a second shaping device is used,
the partially adhered and/or cohered cell paste is transferred from
the first shaping device (e.g., a capillary pipette) to the second
shaping device (e.g., a mold). In further embodiments, the
partially adhered and/or cohered cell paste is transferred by the
first shaping device (e.g., the capillary pipette) into the grooves
of a mold. In still further embodiments, following a maturation
period in which the mold is incubated along with the cell paste
retained therein in a controlled environment to allow the cells in
the cell paste to further adhere and/or cohere to one another to
form the multicellular aggregate, the cohesion of the cells will be
sufficiently strong to allow the resulting multicellular aggregate
to be picked up with an implement (e.g., a capillary pipette). In
still further embodiments, the capillary pipette is suitably be
part of a printing head of a bioprinter or similar apparatus
operable to automatically place the multicellular aggregate into a
three-dimensional construct.
[0156] In some embodiments, the cross-sectional shape and size of
the multicellular aggregates will substantially correspond to the
cross-sectional shapes and sizes of the first shaping device and
optionally the second shaping device used to make the multicellular
aggregates, and the skilled artisan will be able to select suitable
shaping devices having suitable cross-sectional shapes,
cross-sectional areas, diameters, and lengths suitable for creating
multicellular aggregates having the cross-sectional shapes,
cross-sectional areas, diameters, and lengths discussed above.
Placing Cohered Multicellular Aggregates onto a Support
[0157] A number of methods are suitable to place multicellular
aggregates on a support to produce a desired three-dimensional
structure. For example, in some embodiments, the multicellular
aggregates are manually placed in contact with one another,
deposited in place by extrusion from a pipette, nozzle, or needle,
or positioned by an automated, computer-assisted device such as a
bioprinter.
[0158] As described herein, in various embodiments, multicellular
aggregates have many suitable shapes and sizes. In some
embodiments, multicellular aggregates are elongate with any of
several suitable cross-sectional shapes including, by way of
non-limiting example, circular, oval, square, triangular,
polygonal, and irregular. In further embodiments, multicellular
aggregates are elongate and in the form of a cylinder. In some
embodiments, elongate multicellular aggregates are of similar
lengths and/or diameters. In other embodiments, elongate
multicellular aggregates are of differing lengths and/or diameters.
In some embodiments, multicellular aggregates are substantially
spherical. In some embodiments, the engineered tissues (e.g.,
vascular wall segments, etc.) include substantially spherical
multicellular aggregates that are substantially similar in size. In
other embodiments, the engineered tissues (e.g., vascular wall
segments, etc.) include substantially spherical multicellular
aggregates that are of differing sizes. In some embodiments,
engineered tissues (e.g., vascular wall segments, etc.) of
different shapes and sizes are formed by arranging multicellular
aggregates of various shapes and sizes.
[0159] In some embodiments, the cohered multicellular aggregates
are placed onto a support. In various embodiments, the support is
any suitable biocompatible surface. In still further embodiments,
suitable biocompatible surfaces include, by way of non-limiting
examples, polymeric material, porous membranes, plastic, glass,
metal, hydrogel, and combinations thereof. In some embodiments, the
support is coated with a biocompatible substance including, by way
of non-limiting examples, a hydrogel, a protein, a chemical, a
peptide, antibodies, growth factors, or combinations thereof. In
one embodiment, the support is coated with NovoGel.TM.. In another
embodiment, the support is coated with agarose. In one embodiment,
the cohered multicellular aggregates are placed into the wells of a
biocompatible multi-well container.
[0160] Once placement of the cohered multicellular aggregates is
complete, in some embodiments, a tissue culture medium is poured
over the top of the construct. In further embodiments, the tissue
culture medium enters the spaces between the multicellular bodies
to support the cells in the multicellular bodies.
Applying a Layer of a First Type of Cells and/or a Layer of a
Second Type of Cells
[0161] A number of methods are suitable to apply one or more layers
of cells on one or more external surfaces of the cohered mammalian
cell construct. For example, in some embodiments, applying a layer
of cells comprises coating one or more surfaces of said cohered
multicellular aggregates with a suspension, sheet, monolayer, or
fused aggregates of cells. In various embodiments, 1, 2, 3, 4, or
more surfaces of the cohered mammalian cell construct are
coated.
[0162] In some embodiments, applying a layer of cells comprises
bioprinting an additional layer of fused multicellular aggregates.
In other embodiments, applying a layer of cells comprises
bioprinting, spraying, or ink-jetting a solution, suspension, or
liquid concentrate of cells. In further embodiments, a suitable
cell suspension comprises about 1.times.10.sup.4 to about
1.times.10.sup.6 cells/.mu.l. In still further embodiments, a
suitable cell suspension comprises about 1.times.10.sup.5 to about
1.5.times.10.sup.5 cells/.mu.l. In further embodiments, applying a
layer of cells comprises dispensing a suspension of cells directly
onto one or more surfaces of the cohered mammalian cell construct
as spatially-distributed droplets. In still further embodiments,
applying a layer of cells comprises dispensing a suspension of
cells directly onto one or more surfaces of the cohered mammalian
cell construct as a spray. Layers of cells are, in various
embodiments, applied at any suitable time in the construction
process. In some embodiments, one or more layers of cells are
applied on one or more external surfaces of the cohered mammalian
cell construct immediately after bioprinting (e.g., up to 10 min.).
In other embodiments, one or more layers are applied after
bioprinting (e.g., after 10 min.). In yet other embodiments, one or
more layers are applied during maturation of the construct.
[0163] Any type of cell is suitable for application as a layer by
bioprinting as bio-ink. Moreover, any type of cell is suitable for
application as a layer by deposition as droplets of suspension,
solution, or concentrate, or spraying as a suspension, solution, or
concentrate. In some embodiments, fibroblasts are applied as one or
more layers of cells on one or more external surfaces of the
cohered mammalian cell construct. In other embodiments, endothelial
cells are applied as one or more layers of cells on one or more
external surfaces of the cohered mammalian cell construct. In
further embodiments, a layer of endothelial cells is applied to one
or more external surfaces of the cohered mammalian cell construct
and a layer of fibroblasts is applied to one or more distinct
surfaces of the construct.
[0164] Example 9 demonstrates vascular wall constructs bioprinted
with cohered smooth muscle cell aggregates, which were further
coated with an endothelial cell concentrate (e.g.,
1-1.5.times.10.sup.5 cells/.mu.l). The techniques of Example 9
resulted in a vascular wall construct comprised of SMC and a
covering of EC (e.g., a putative tunica media and tunica intima).
See, e.g., FIGS. 3, 4B.
[0165] Example 10 demonstrates vascular wall constructs bioprinted
with cohered human aortic smooth muscle cell aggregates. Further,
human aortic endothelial cells in suspension were dispensed from a
bioprinter on top of the smooth muscle cylindrical bio-ink as 2.5
.mu.L droplets.
[0166] In some embodiments, the methods further comprise the step
of culturing a layer of cells on a support. In such embodiments,
applying a layer of cells, in some cases, comprises placing one or
more surfaces of the cohered smooth muscle cell construct in direct
contact with an established culture of cells. In further
embodiments, the construct is bioprinted directly onto a cultured
layer of cells or a monolayer of cells. Any type of cultured cell
layer on a biocompatible support is suitable. In some embodiments,
multicellular aggregates are bioprinted onto a layer of endothelial
cells. In other embodiments, multicellular aggregates are
bioprinted onto a layer of fibroblasts. In further embodiments, the
layer of cells adheres and/or coheres with the multicellular
aggregates of the bioprinted construct. In some embodiments, each
layer of a multi-layered structure are bioprinted. In further
embodiments, the individual layers comprise variable forms of
bio-ink, including but not limited to: cohered cell aggregates,
cell paste, cell paste in combination with extrusion compound(s) or
other additives, cell monolayers, and cell sheets.
[0167] Example 11 demonstrates construction of the same constructs
of Example 10; however, the constructs were bioprinted onto a
support on which a confluent monolayer of human dermal fibroblasts
had been pre-cultured. The techniques of Example 11 resulted in a
vascular wall construct comprised of SMC and coverings of EC and Fb
(e.g., a putative tunica media, tunica intima, and tunica
adventitia). See, e.g., FIGS. 4a and 4b.
Incubating Multicellular Aggregates
[0168] In some embodiments, the multicellular aggregates are
incubated. In further embodiments, the incubation allows the
multicellular aggregates adhere and/or cohere to form a tissue,
such as a vascular wall segment. In some embodiments, the
multicellular aggregates cohere to form a tissue in a cell culture
environment (e.g., a Petri dish, cell culture flask, bioreactor,
etc.). In further embodiments, the multicellular aggregates cohere
to form a tissue in an environment with conditions suitable to
facilitate growth of the cell types included in the multicellular
aggregates. In one embodiment, the multicellular aggregates are
incubated at about 37.degree. C., in a humidified atmosphere
containing about 5% CO.sub.2, in the presence of cell culture
medium containing factors and/or ions to foster adherence and/or
coherence. In other embodiments, the multicellular aggregates are
maintained in an environment that contains 0.1% to 21% O.sub.2.
[0169] The incubation, in various embodiments, has any suitable
duration. In further various embodiments, the incubation has a
duration of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150, 160, 170, 180, or more minutes, including increments
therein. In further various embodiments, the incubation has a
duration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, or more hours,
including increments therein. In further various embodiments, the
incubation has a duration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days, including
increments therein. Several factors influence the time required for
multicellular aggregates to cohere to form a tissue including, by
way of non-limiting examples, cell types, cell type ratios, culture
conditions, and the presence of additives such as growth
factors.
Additional Steps for Increasing Viability of the Engineered
Tissue
[0170] In some embodiments, the method further comprises steps for
increasing the viability of the engineered tissue. In further
embodiments, these steps involve providing direct contact between
the tissue and a nutrient medium through a temporary or
semi-permanent lattice of confinement material. In some
embodiments, the tissue is constrained in a porous or gapped
material. Direct access of at least some of the cells of the
engineered tissue to nutrients increases the viability of the
engineered tissue.
[0171] In further embodiments, the additional and optional steps
for increasing the viability of the engineered tissue include: 1)
optionally dispensing base layer of confinement material prior to
placing cohered multicellular aggregates; 2) optionally dispensing
a perimeter of confinement material; 3) bioprinting cells of the
tissue within a defined geometry; and 4) dispensing elongate bodies
(e.g., cylinders, ribbons, etc.) of confinement material overlaying
the nascent tissue in a pattern that introduces gaps in the
confinement material, such as a lattice, mesh, or grid. See, e.g.,
Example 12 and FIG. 5.
[0172] Many confinement materials are suitable for use in the
methods described herein. In some embodiments, hydrogels are
exemplary confinement materials possessing one or more advantageous
properties including: non-adherent, biocompatible, extrudable,
bioprintable, non-cellular, of suitable strength, and not soluble
in aqueous conditions. In some embodiments, suitable hydrogels are
natural polymers. In one embodiment, the confinement material is
comprised of NovoGel.TM.. In further embodiments, suitable
hydrogels include those derived from surfactant polyols such as
Pluronic F-127, collagen, hyaluronate, fibrin, alginate, agarose,
chitosan, and derivatives or combinations thereof. In other
embodiments, suitable hydrogels are synthetic polymers. In further
embodiments, suitable hydrogels include those derived from
poly(acrylic acid) and derivatives thereof, poly(ethylene oxide)
and copolymers thereof, poly(vinyl alcohol), polyphosphazene, and
combinations thereof. In various specific embodiments, the
confinement material is selected from: hydrogel, NovoGel.TM.,
agarose, alginate, gelatin, Matrigel.TM., hyaluronan, poloxamer,
peptide hydrogel, poly(isopropyl n-polyacrylamide), polyethylene
glycol diacrylate (PEG-DA), hydroxyethyl methacrylate,
polydimethylsiloxane, polyacrylamide, poly(lactic acid), silicon,
silk, and combinations thereof.
[0173] In some embodiments, the gaps overlaying pattern are
distributed uniformly or substantially uniformly around the surface
of the tissue. In other embodiments, the gaps are distributed
non-uniformly, whereby the cells of the tissue are exposed to
nutrients non-uniformly. In non-uniform embodiments, the
differential access to nutrients is optionally exploited to
influence one or more properties of the tissue. For instance, in
some cases, it is desirable to have cells on one surface of a
bioprinted tissue proliferate faster than cells on another surface
of the bioprinted tissue. In some embodiments, the exposure of
various parts of the tissue to nutrients is changed at various
times to influence the development of the tissue toward a desired
endpoint.
[0174] In some embodiments, the confinement material is removed at
any suitable time, including but not limited to, immediately after
bioprinting (e.g., within 10 minutes), after bioprinting (e.g.,
after 10 minutes), before the cells are substantially cohered to
each other, after the cells are substantially cohered to each
other, before the cells produce an extracellular matrix, after the
cells produce an extracellular matrix, just prior to use, and the
like. In various embodiments, confinement material is removed by
any suitable method. For example, in some embodiments, the
confinement material is excised, pulled off the cells, digested, or
dissolved.
[0175] In some embodiments, the methods further comprise the step
of subjecting the engineered tissue (e.g., vascular wall segment,
etc.) to shear force, caused by fluid flow, on one or more
sides.
Particular Exemplary Embodiments
[0176] In certain embodiments, disclosed herein are living,
three-dimensional tissues wherein at least one component of said
tissue was bioprinted; and wherein said tissue is not a vascular
tube. In some embodiments, the tissue is substantially free of any
pre-formed scaffold. In some embodiments, the tissue is
substantially free of any pre-formed scaffold at the time of
bioprinting. In some embodiments, the tissue is substantially free
of any pre-formed scaffold at the time of use. In some embodiments,
at least one component of the tissue comprises a laminar or planar
geometry. In some embodiments, the tissue is secured to a
biocompatible surface on one or more sides. In further embodiments,
the biocompatible surface is a porous membrane. In further
embodiments, the tissue is subjected to shear force, caused by
fluid flow, on one or more sides. In some embodiments, the tissue
is at least about 25 .mu.m in its smallest dimension at the time of
bioprinting. In further embodiments, the tissue is at least about
100 .mu.m in its smallest dimension at the time of bioprinting. In
still further embodiments, the tissue is at least about 250 .mu.m
in its smallest dimension at the time of bioprinting. In still
further embodiments, the tissue is at least about 500 .mu.m in its
smallest dimension at the time of bioprinting. In some embodiments,
the tissue is less than 3.0 cm in its largest dimension at the time
of bioprinting. In some embodiments, the tissue comprises smooth
muscle cells and endothelial cells, wherein the ratio of smooth
muscle cells to endothelial cells is about 90:10 to about 60:40. In
some embodiments, the tissue comprises smooth muscle cells and
endothelial cells, wherein the ratio of smooth muscle cells to
endothelial cells is about 85:15. In some embodiments, the tissue
comprises smooth muscle cells and endothelial cells, wherein the
ratio of smooth muscle cells to endothelial cells is about 70:30.
In some embodiments, the tissue comprises smooth muscle cells and
fibroblasts, wherein the ration of smooth muscle cells to
fibroblasts is about 90:10 to about 60:40. In some embodiments, the
tissue comprises smooth muscle cells, fibroblasts, and endothelial
cells, wherein the ratio of smooth muscle cells to fibroblasts and
endothelial cells is about 70:25:5. In some embodiments, the tissue
is for use in in vitro assays. In further embodiments, the tissue
is for use in drug testing. In still further embodiments, the
tissue is for use in cardiovascular drug testing. In some
embodiments, the smooth muscle cells, fibroblasts, and endothelial
cells are adult, differentiated cells. In some embodiments, the
smooth muscle cells, fibroblasts, and endothelial cells are adult,
non-differentiated cells. In some embodiments, the smooth muscle
cells are human smooth muscle cells. In further embodiments, the
smooth muscle cells originated from a tissue selected from the
group consisting of: blood, blood vessel, lymphatic vessel, tissue
of the digestive tract, tissue of the genitourinary tract, adipose
tissue, tissue of the respiratory tract, tissue of the reproductive
system, mesoderm-derived tissue, bone marrow, and umbilical tissue.
In some embodiments, the endothelial cells are human endothelial
cells. In further embodiments, the endothelial cells originated
from a tissue selected from the group consisting of: blood, blood
vessel, lymphatic vessel, tissue of the digestive tract, tissue of
the genitourinary tract, adipose tissue, tissue of the respiratory
tract, tissue of the reproductive system, mesoderm-derived tissue,
bone marrow, and umbilical tissue. In some embodiments, the
fibroblasts are non-vascular fibroblasts. In other embodiments, the
fibroblasts are derived from the vascular adventitia. In some
embodiments, one or more of said cell types are derived from a
particular vertebrate subject. In further embodiments, the cells
are derived from a vertebrate subject that has a disease or
condition that affects the cardiovascular system. In some
embodiments, the cells are selected to mimic a particular disease
state. In some embodiments, the cells are configured to mimic a
particular disease state. In some embodiments, the cells are
treated or modulated in a manner that mimics a particular disease
state.
[0177] In certain embodiments, disclosed herein are arrays of
living, three-dimensional, tissues, wherein each said tissue
comprises one or more types of mammalian cells; wherein said cells
are cohered to one another; wherein at least one component of each
said tissue was bioprinted; and wherein each said tissue is
maintained in culture. In some embodiments, each tissue within the
array is free of any pre-formed scaffold at the time of use. In
some embodiments, the mammalian cells are selected from the group
consisting of: liver cells, gastrointestinal cells, pancreatic
cells, kidney cells, lung cells, tracheal cells, vascular cells,
skeletal muscle cells, cardiac cells, skin cells, smooth muscle
cells, connective tissue cells, corneal cells, genitourinary cells,
reproductive cells, endothelial cells, epithelial cells,
fibroblasts, neural cells, Schwann cells, adipose cells, bone
cells, bone marrow cells, cartilage cells, pericytes, mesenchymal
cells, mesothelial cells, stromal cells, stem cells, progenitor
cells, lymph cells, blood cells, tumor-derived cells, and
combinations thereof. In some embodiments, each tissue within the
array is substantially similar. In other embodiments, one or more
of the tissues within the array is unique. In some embodiments,
individual tissues within the array represent one or more specific
tissues in the human body. In further embodiments, one or more
individual tissues within the array represent human tissues
selected from the group consisting of: blood or lymph vessel,
muscle, uterus, nerve, mucous membrane, mesothelium, omentum,
cornea, skin, liver, kidney, heart, trachea, lung, bone, bone
marrow, adipose, connective, bladder, breast, pancreas, spleen,
brain, esophagus, stomach, intestine, colon, rectum, ovary, and
prostate; wherein each of the tissues optionally incorporates
compositional or architectural features of specific disease states
(e.g., fibrosis, cancer, inflammation, etc.). In some embodiments,
each tissue exists in a well of a biocompatible multi-well
container. In further embodiments, the wells are coated with one of
or more of the following: a biocompatible hydrogel, a protein, a
chemical, a peptide, antibodies, or growth factors. In still
further embodiments, the wells are coated with agarose. In some
embodiments, each tissue was placed onto a porous, biocompatible
membrane within said wells of said container. In some embodiments,
the container is compatible with automated drug screening. In some
embodiments, each tissue is affixed to a biocompatible surface on
one or more sides. In further embodiments, the biocompatible
surface is a porous membrane. In still further embodiments, each
tissue is subjected to shear force, caused by fluid flow, on one or
more sides. In some embodiments, the tissues within the array are
generated with one or more cell types derived from two or more
distinct human donors. In some embodiments, each tissue within the
array is maintained independently in culture. In other embodiments,
two or more individual tissues within the array exchange soluble
factors. In some embodiments, the array is for use in in vitro
assays. In further embodiments, the array is for use in drug
testing.
[0178] In certain embodiments, disclosed herein are methods of
constructing an array of living, three-dimensional mammalian
tissues comprising the steps of: preparing cohered multicellular
aggregates comprising mammalian cells; placing said cohered
multicellular aggregates onto a biocompatible support; wherein said
aggregates are spatially arranged in a form suitable for a tissue
array; and incubating said multicellular aggregates to allow them
to cohere and form an array of three-dimensional tissues; wherein
said incubation has a duration of about 2 hours to about 10 days.
In some embodiments, at least one component of each tissue within
the array was bioprinted. In further embodiments, each tissue
within the array is substantially free of any pre-formed scaffold
at the time of use. In various embodiments, the array comprises
from 2 to 500 distinct tissues. In further embodiments, the tissues
are spatially arranged in a defined pattern. In still further
embodiments, the tissues are arranged in a grid of rows and
columns. In some embodiments, the cohered multicellular aggregates
comprise one cell type. In other embodiments, the cohered
multicellular aggregates comprise more than one cell type. In some
embodiments, the cohered multicellular aggregates are substantially
spherical and/or substantially cylindrical. In some embodiments,
the biocompatible support consists of: a polymeric material, a
porous membrane, plastic, glass, metal, or hydrogel. In some
embodiments, each tissue within the array is at least about 25
.mu.m in its smallest dimension at the time of bioprinting. In
further embodiments, each tissue is at least about 150 .mu.m in its
smallest dimension at the time of bioprinting. In still further
embodiments, each tissue is at least about 250 .mu.m in its
smallest dimension at the time of bioprinting. In still further
embodiments, each tissue is at least about 500 .mu.m in its
smallest dimension at the time of bioprinting. In some embodiments,
each tissue within the array is maintained in culture. In some
embodiments, the methods further comprise the step of subjecting
each said tissue to shear force, caused by fluid flow, on one or
more sides.
[0179] In certain embodiments, disclosed herein are living,
three-dimensional vascular wall segments comprising: smooth muscle
cells; and optionally, one or more cell types selected from the
group consisting of: fibroblasts and endothelial cells; wherein
said cells are cohered to one another; wherein at least one
component of said vascular wall segment was bioprinted; and wherein
said vascular wall segment is non-tubular. In some embodiments, the
vascular wall segment is free of any pre-formed scaffold. In some
embodiments, the vascular wall segment is substantially planar. In
some embodiments, the vascular wall segment is affixed to a
biocompatible surface on one or more sides. In further embodiments,
the biocompatible surface is a porous membrane. In still further
embodiments, the vascular wall segment is subjected to shear force,
caused by fluid flow, on one or more sides. In some embodiments,
the vascular wall segment is at least about 25 .mu.m in its
smallest dimension at the time of bioprinting. In further
embodiments, the vascular wall segment is at least about 150 .mu.m
in its smallest dimension at the time of bioprinting. In still
further embodiments, the vascular wall segment is at least about
250 .mu.m in its smallest dimension at the time of bioprinting. In
still further embodiments, the vascular wall segment is at least
about 500 .mu.m in its smallest dimension at the time of
bioprinting. In some embodiments, the vascular wall segment
comprises smooth muscle cells and endothelial cells, wherein the
ratio of smooth muscle cells to endothelial cells is about 90:10 to
about 60:40. In further embodiments, the vascular wall segment
comprises smooth muscle cells and endothelial cells, wherein the
ratio of smooth muscle cells to endothelial cells is about 85:15.
In other embodiments, the vascular wall segment comprises smooth
muscle cells and endothelial cells, wherein the ratio of smooth
muscle cells to endothelial cells is about 70:30. In some
embodiments, the vascular wall segment comprises smooth muscle
cells and fibroblasts, wherein the ration of smooth muscle cells to
fibroblasts is about 90:10 to about 60:40. In some embodiments, the
vascular wall segment comprises smooth muscle cells, fibroblasts,
and endothelial cells, wherein the ratio of smooth muscle cells to
fibroblasts and endothelial cells is about 70:25:5. In some
embodiments, the vascular wall segment is for use in in vitro
assays. In further embodiments, the vascular wall segment is for
use in drug testing. In still further embodiments, the vascular
wall segment is for use in cardiovascular drug testing. In some
embodiments, the smooth muscle cells, fibroblasts, and endothelial
cells are adult, differentiated cells. In other embodiments, the
smooth muscle cells, fibroblasts, and endothelial cells are adult,
non-differentiated cells. In some embodiments, the smooth muscle
cells are human smooth muscle cells. In further embodiments, the
smooth muscle cells originated from a tissue selected from the
group consisting of: blood, blood vessel, lymphatic vessel, tissue
of the digestive tract, tissue of the genitourinary tract, adipose
tissue, tissue of the respiratory tract, tissue of the reproductive
system, bone marrow, and umbilical tissue. In some embodiments, the
endothelial cells are human endothelial cells. In further
embodiments, the endothelial cells originated from a tissue
selected from the group consisting of: blood, blood vessel,
lymphatic vessel, tissue of the digestive tract, tissue of the
genitourinary tract, adipose tissue, tissue of the respiratory
tract, tissue of the reproductive system, bone marrow, and
umbilical tissue. In some embodiments, the fibroblasts are
non-vascular fibroblasts. In some embodiments, the fibroblasts are
derived from the vascular adventitia. In some embodiments, the
cells are derived from a particular vertebrate subject. In further
embodiments, one or more of the cell types are derived from a
vertebrate subject that has a disease or condition that affects the
cardiovascular system. In some embodiments, the cells are selected
to mimic a particular disease state. In some embodiments, the cells
are configured to mimic a particular disease state. In some
embodiments, the cells are treated or modulated in a manner that
mimics a particular disease state.
[0180] In certain embodiments, disclosed herein are arrays of
living, three-dimensional, vascular wall segments, wherein each
said vascular wall segment comprises smooth muscle cells, and
optionally, one or more cell types selected from the group
consisting of: fibroblasts and endothelial cells; wherein said
cells are cohered to one another; wherein each said vascular wall
segment is engineered. In some embodiments, at least one component
of each vascular wall segment within the array was bioprinted. In
further embodiments, each vascular wall segment within the array is
free of any pre-formed scaffold at the time of use. In some
embodiments, each vascular wall segment exists within a well of a
biocompatible multi-well container. In further embodiments, the
wells are coated with one of or more of the following: a
biocompatible hydrogel, a protein, a chemical, a peptide,
antibodies, or growth factors. In still further embodiments, the
wells are coated with NovoGel.TM.. In other embodiments, the wells
are coated with agarose. In some embodiments, each vascular wall
segment was placed onto a porous, biocompatible membrane within
said wells of said container. In further embodiments, the container
is compatible with automated drug screening. In some embodiments,
each vascular wall segment is affixed to a biocompatible surface on
one or more sides. In further embodiments, the biocompatible
surface is a porous membrane. In still further embodiments, each
vascular wall segment is subjected to shear force, caused by fluid
flow, on one or more sides. In some embodiments, each vascular wall
segment within the array is substantially similar. In other
embodiments, one or more of the vascular wall segments within the
array are unique. In some embodiments, the vascular wall segments
within the array represent one or more distinct vascular tissues in
the human body. In some embodiments, the vascular wall segments
within the array are generated with one or more cell types derived
from two or more distinct human donors. In some embodiments, each
vascular wall segment within the array is maintained independently
in culture. In other embodiments, two or more individual vascular
wall segments within the array exchange soluble factors. In some
embodiments, the array is for use in in vitro assays. In further
embodiments, the array is for use in drug testing. In still further
embodiments, the array is for use in cardiovascular drug
testing.
[0181] In certain embodiments, disclosed herein are methods of
constructing a living, three-dimensional vascular wall segment
comprising the steps of: preparing cohered multicellular aggregates
comprising smooth muscle cells; placing said cohered multicellular
aggregates onto a support; and incubating said multicellular
aggregates to allow them to cohere and form a vascular wall
segment; wherein said incubation has a duration of about 2 hours to
about 10 days. In some embodiments, at least one component of the
vascular wall segment was bioprinted. In further embodiments, the
vascular wall segment is used in in vitro assays and is free of any
pre-formed scaffold at the time of use. In some embodiments, the
cohered multicellular aggregates further comprise endothelial
cells. In some embodiments, the cohered multicellular aggregates
further comprise fibroblasts. In some embodiments, the cohered
multicellular aggregates are substantially spherical or
substantially cylindrical. In some embodiments, the cohered
multicellular aggregates are placed onto a biocompatible surface.
In further embodiments, the biocompatible surface consists of: a
polymeric material, a porous membrane, plastic, glass, metal, or
hydrogel. In some embodiments, the vascular wall segment is at
least about 50 .mu.m in its smallest dimension at the time of
bioprinting. In further embodiments, the vascular wall segment is
at least about 150 .mu.m in its smallest dimension at the time of
bioprinting. In still further embodiments, the vascular wall
segment is at least about 266 .mu.m in its smallest dimension at
the time of bioprinting. In other embodiments, the vascular wall
segment is at least about 500 .mu.m in its smallest dimension at
the time of bioprinting. In some embodiments, the method further
comprises the step of subjecting said vascular wall segment to
shear force, caused by fluid flow, on one or more sides.
[0182] In certain embodiments, disclosed herein are living,
three-dimensional tissues comprising: smooth muscle cells, wherein
said smooth muscle cells are cohered to one another; and one or
more of: a layer of endothelial cells on one or more surfaces; a
layer of fibroblasts on one or more surfaces; wherein at least one
component of said tissue was bioprinted; and wherein said tissue is
non-tubular. In some embodiments, the tissue is substantially free
of any pre-formed scaffold. In some embodiments, the tissue is
substantially free of any pre-formed scaffold at the time of
bioprinting. In some embodiments, the tissue is substantially free
of any pre-formed scaffold at the time of use. In some embodiments,
the tissue is substantially planar. In some embodiments, the layer
of endothelial cells comprises a monolayer, one or more sheets, or
fused aggregates of endothelial cells. In some embodiments, the
tissue comprises a layer of endothelial cells on one or more
surfaces of said tissue. In some embodiments, the layer of
fibroblasts comprises a monolayer, one or more sheets, or fused
aggregates of fibroblasts. In some embodiments, the tissue
comprises a layer of fibroblasts on one or more surfaces of said
tissue. In some embodiments, the tissue comprises a layer of
endothelial cells and a layer of fibroblasts; wherein said layer of
endothelial cells is on one or more external surfaces of said
tissue and said layer of fibroblasts is one or more distinct
surfaces of said tissue. In some embodiments, the tissue is at
least about 50 .mu.m in its smallest dimension at the time of
bioprinting. In further embodiments, the tissue is at least about
150 .mu.m in its smallest dimension at the time of bioprinting. In
still further embodiments, the tissue is at least about 250 .mu.m
in its smallest dimension at the time of bioprinting. In still
further embodiments, the tissue is at least about 500 .mu.m in its
smallest dimension at the time of bioprinting. In some embodiments,
the tissue is affixed to a biocompatible surface on one or more
sides. In further embodiments, the biocompatible surface is a
porous membrane. In further embodiments, the tissue is subjected to
shear force, caused by fluid flow, on one or more sides. In some
embodiments, the tissue is for use in in vitro assays. In further
embodiments, the tissue is for use in drug testing. In still
further embodiments, the tissue is for use in cardiovascular drug
testing. In some embodiments, the smooth muscle cells, fibroblasts,
and endothelial cells are adult, differentiated cells. In some
embodiments, the smooth muscle cells, fibroblasts, and endothelial
cells are adult, non-differentiated cells. In some embodiments, the
smooth muscle cells are human smooth muscle cells. In further
embodiments, the smooth muscle cells originated from a tissue
selected from the group consisting of: blood, vascular tissue,
blood vessel, lymphatic vessel, tissue of the digestive tract,
tissue of the genitourinary tract, adipose tissue, tissue of the
respiratory tract, tissue of the reproductive system, bone marrow,
muscle tissue, connective tissue, mesoderm-derived tissue, and
umbilical tissue. In some embodiments, the endothelial cells are
human endothelial cells. In further embodiments, the endothelial
cells originated from a tissue selected from the group consisting
of: vascular tissue, blood, blood vessel, lymphatic vessel, tissue
of the digestive tract, tissue of the genitourinary tract, adipose
tissue, tissue of the respiratory tract, tissue of the reproductive
system, mesoderm-derived tissue, bone marrow, and umbilical tissue.
In some embodiments, the fibroblasts are non-vascular fibroblasts.
In other embodiments, the fibroblasts are derived from the vascular
adventitia. In some embodiments, the cells are derived from a
particular vertebrate subject. In further embodiments, the cells
are derived from a vertebrate subject that has a disease or
condition that affects the cardiovascular system. In some
embodiments, the cells are selected to mimic a particular disease
state. In some embodiments, the cells are configured to mimic a
particular disease state. In some embodiments, the cells are
treated or modulated in a manner that mimics a particular disease
state.
[0183] In certain embodiments, disclosed herein are arrays of
living, three-dimensional, tissues, wherein each said tissue
comprises mammalian cells, wherein said cells are cohered to one
another; and one or more of: a layer of a first type of mammalian
cells on one or more surfaces; a layer of a second type of
mammalian cells on one or more surfaces; wherein at least one
component of each said tissue was bioprinted; wherein each said
tissue is maintained in culture. In some embodiments, each tissue
within the array is free of any pre-formed scaffold at the time of
use. In some embodiments, the mammalian cells include smooth muscle
cells derived from a tissue selected from the group consisting of:
vascular tissue, blood, blood vessel, lymphatic vessel, tissue of
the digestive tract, tissue of the genitourinary tract, adipose
tissue, tissue of the respiratory tract, tissue of the reproductive
system, bone marrow, muscle tissue, mesenchymal tissue, connective
tissue, mesoderm-derived tissue, and umbilical tissue. In some
embodiments, the mammalian cells include endothelial cells derived
from a tissue selected from the group consisting of: vascular
tissue, blood, blood vessel, lymphatic vessel, tissue of the
digestive tract, tissue of the genitourinary tract, adipose tissue,
tissue of the respiratory tract, tissue of the reproductive system,
mesoderm-derived tissue, bone marrow, and umbilical tissue. In some
embodiments, the said mammalian cells include non-vascular
fibroblasts. In other embodiments, the mammalian cells include
vascular fibroblasts. In further embodiments, the vascular
fibroblasts are derived from vascular adventitia. In some
embodiments, each tissue within the array is substantially similar.
In other embodiments, one or more of the tissues within the array
is unique. In some embodiments, individual tissues within the array
represent one or more specific tissues in the human body. In
further embodiments, one or more individual tissues within the
array represent human tissues selected from the group consisting
of: blood or lymph vessel, muscle, uterus, nerve, mucous membrane,
mesothelium, omentum, cornea, skin, liver, kidney, heart, trachea,
lung, bone, bone marrow, adipose, connective, bladder, breast,
pancreas, spleen, brain, esophagus, stomach, intestine, colon,
rectum, ovary, and prostate. In some embodiments, each tissue
exists in a well of a biocompatible multi-well container. In
further embodiments, the wells are coated with one of or more of
the following: a biocompatible hydrogel, a protein, a chemical, a
peptide, antibodies, or growth factors. In some embodiments, the
wells are coated with NovoGel.TM.. In still further embodiments,
the wells are coated with agarose. In some embodiments, each tissue
was placed onto a porous, biocompatible membrane within the wells
of the container. In some embodiments, the container is compatible
with automated drug screening. In some embodiments, each tissue
within the array is affixed to a biocompatible surface on one or
more sides. In further embodiments, the biocompatible surface is a
porous membrane. In still further embodiments, each tissue is
subjected to shear force, caused by fluid flow, on one or more
sides. In some embodiments, the tissues within the array are
generated with one or more cell types derived from two or more
distinct human donors. In some embodiments, each tissue within the
array is maintained independently in culture. In other embodiments,
two or more individual tissues within the array exchange soluble
factors. In some embodiments, the array is for use in in vitro
assays. In further embodiments, the array is for use in drug
testing.
[0184] In certain embodiments, disclosed herein are methods of
constructing a living, three-dimensional tissue comprising the
steps of: preparing one or more cohered multicellular aggregates
comprising mammalian cells; placing said one or more cohered
multicellular aggregates onto a support; applying, to said one or
more cohered multicellular aggregates, one or more of: a layer of a
first type of mammalian cells on one or more external surfaces; a
layer of a second type of mammalian cells on one or more external
surfaces; and incubating said one or more multicellular aggregates
to allow them to cohere and to form a tissue; wherein said
incubation has a duration of about 2 hours to about 10 days. In
some embodiments, at least one component of said tissue was
bioprinted. In some embodiments, the tissue is free of any
pre-formed scaffold at the time of manufacture. In further
embodiments, the tissue is substantially free of any pre-formed
scaffold at the time of manufacture. In other embodiments, the
tissue is substantially free of any pre-formed scaffold at the time
of use. In some embodiments, the tissue is at least about 50 .mu.m
in its smallest dimension at the time of bioprinting. In further
embodiments, the tissue is at least about 150 .mu.m in its smallest
dimension at the time of bioprinting. In still further embodiments,
the tissue is at least about 250 .mu.m in its smallest dimension at
the time of bioprinting. In other embodiments, the tissue is at
least about 500 .mu.m in its smallest dimension at the time of
bioprinting. In further embodiments, the tissue has a length,
width, or height, or thickness of about 50 .mu.m to about 600 .mu.m
in the smallest dimension. In still further embodiments the tissue
has a length, width, height, or thickness greater than 1 mm. In
some embodiments, the cohered multicellular aggregates of the first
cell type comprise stromal cells, connective tissue-derived cells,
cells that are mesodermal in origin. In further embodiments, the
cohered multicellular aggregates additionally comprise second cell
types. In additional embodiments, the second cell type(s) are
derived from epithelial tissues, endothelial tissues, mesenchymal
tissues, or ectodermal tissues. In some embodiments, applying a
layer of mammalian cells comprises coating at least one surface of
the cohered multicellular aggregates with a suspension, a
monolayer, one or more sheets, multiple layers, or fused aggregates
of cells. In further embodiments, the suspension comprises about
1.times.10.sup.4 to about 1.times.10.sup.6 cells/.mu.l. In still
further embodiments, the suspension comprises about
1.times.10.sup.5 to about 1.5.times.10.sup.5 cells/.mu.l. In some
embodiments, applying a layer of mammalian cells comprises
dispensing a suspension of cells directly onto one surface of said
cohered multicellular aggregates as spatially-distributed droplets.
In some embodiments, applying a layer of mammalian cells comprises
dispensing a suspension of cells directly onto one surface of said
cohered multicellular aggregates as a spray. In some embodiments,
applying a layer of mammalian cells comprises placing one or more
surfaces of said cohered multicellular aggregates in direct contact
with an established layer of cells. In further embodiments, the
established layer of cells comprises a monolayer, multiple layers,
one or more sheets, or fused aggregates of cells. In some
embodiments, a layer of a first type of cells is applied on one or
more surfaces of said cohered multicellular aggregates and a layer
of a second type of cells is applied to one or more distinct
surfaces of said cohered multicellular aggregates. In some
embodiments, the incubation has a duration of about 2 hours to
about 10 days. In some embodiments, the step of applying one or
more of: a layer of a first type of cells on one or more surfaces;
a layer of a second type of cells on one or more surfaces is
performed at the time the one or more cohered multicellular
aggregates are placed. In other embodiments, the step of applying
one or more of: a layer of a first type of cells on one or more
external surfaces; a layer of a second type of cells on one or more
external surfaces is performed during said incubation. In some
embodiments, the methods further comprise the step of subjecting
the tissue to shear force, caused by fluid flow, on one or more
sides.
[0185] In certain embodiments, disclosed herein are living,
three-dimensional vascular wall segments comprising: smooth muscle
cells, wherein said smooth muscle cells are cohered to one another;
and one or more of: a layer of endothelial cells on one or more
surfaces; a layer of fibroblasts on one or more surfaces; wherein
at least one component of said vascular wall segment was
bioprinted; and wherein said vascular wall segment is non-tubular.
In some embodiments, the vascular wall segment is substantially
free of any pre-formed scaffold at the time of manufacture. In
other embodiments, the vascular wall segment is substantially free
of any pre-formed scaffold at the time of use. In some embodiments,
the vascular wall segment is substantially planar. In some
embodiments, the layer of endothelial cells comprises a monolayer,
one or more layers, one or more sheets, or fused aggregates of
endothelial cells. In some embodiments, the vascular wall segment
comprises a layer of endothelial cells on one or more surfaces. In
some embodiments, the layer of fibroblasts comprises a monolayer,
one or more layers, one or more sheets, or fused aggregates of
fibroblasts. In some embodiments, the vascular wall segment
comprises a layer of fibroblasts on one or more surfaces of said
vascular wall segment. In some embodiments, the vascular wall
segment comprises a layer of endothelial cells and said layer of
fibroblasts; wherein said layer of endothelial cells is on one or
more external surfaces of said vascular wall segment and said layer
of fibroblasts is one or more distinct surfaces of said vascular
wall segment. In some embodiments, the vascular wall segment is at
least about 50 .mu.m in its smallest dimension at the time of
bioprinting. In further embodiments, the vascular wall segment is
at least about 150 .mu.m in its smallest dimension at the time of
bioprinting. In still further embodiments, the vascular wall
segment is at least about 250 .mu.m in its smallest dimension at
the time of bioprinting. In still further embodiments, the vascular
wall segment is at least about 500 .mu.m in its smallest dimension
at the time of bioprinting. In some embodiments, the vascular wall
segment is affixed to a biocompatible surface on one or more sides.
In further embodiments, the biocompatible surface is a porous
membrane. In still further embodiments, the vascular wall segment
is subjected to shear force, caused by fluid flow, on one or more
sides. In some embodiments, the vascular wall segment is for use in
in vitro assays. In further embodiments, the vascular wall segment
is for use in drug testing. In still further embodiments, the
vascular wall segment is for use in cardiovascular drug testing. In
some embodiments, the smooth muscle cells, fibroblasts, and
endothelial cells are adult, differentiated cells. In other
embodiments, the smooth muscle cells, fibroblasts, and endothelial
cells are adult, non-differentiated cells. In some embodiments, the
smooth muscle cells are human smooth muscle cells. In further
embodiments, the smooth muscle cells originated from a tissue
selected from the group consisting of: vascular tissue, blood,
blood vessel, lymphatic vessel, tissue of the digestive tract,
tissue of the genitourinary tract, adipose tissue, tissue of the
respiratory tract, tissue of the reproductive system, bone marrow,
muscle tissue, connective tissue, and umbilical tissue. In some
embodiments, the endothelial cells are human endothelial cells. In
further embodiments, the endothelial cells originated from a tissue
selected from the group consisting of: vascular tissue, blood,
blood vessel, lymphatic vessel, tissue of the digestive tract,
tissue of the genitourinary tract, adipose tissue, tissue of the
respiratory tract, tissue of the reproductive system, bone marrow,
and umbilical tissue. In some embodiments, the fibroblasts are
non-vascular fibroblasts. In other embodiments, the fibroblasts are
vascular fibroblasts. In further embodiments, the fibroblasts are
derived from the vascular adventitia. In some embodiments, one or
more of the cellular components are derived from a particular
vertebrate subject. In further embodiments, one or more of the
cellular components are derived from a vertebrate subject that has
a disease or condition that affects the cardiovascular system. In
some embodiments, one or more of the cellular components are
selected and/or configured to mimic a particular disease state. In
some embodiments, one or more of the cellular components are
treated and/or modulated in a manner that mimics a particular
disease state.
[0186] In certain embodiments, disclosed herein are arrays of
living, three-dimensional vascular wall segments, wherein each said
vascular wall segment comprises smooth muscle cells, wherein said
smooth muscle cells are cohered to one another; and one or more of:
a layer of endothelial cells on one or more surfaces; a layer of
fibroblasts on one or more surfaces; wherein each said vascular
wall segment is engineered; wherein each said vascular wall segment
is maintained in culture. In some embodiments, at least one
component of each vascular wall segment within the array was
bioprinted. In further embodiments, each vascular wall segment
within the array is substantially free of any pre-formed scaffold
at the time of manufacture. In other embodiments, each vascular
wall segment within the array is substantially free of any
pre-formed scaffold at the time of use. In some embodiments, each
vascular wall segment exists within a well of a biocompatible
multi-well container. In further embodiments, the wells are coated
with one of or more of the following: a biocompatible hydrogel, a
protein, a chemical, a peptide, antibodies, or growth factors. In
some embodiments, the wells are coated with NovoGel.TM.. In other
embodiments, the wells are coated with agarose. In some
embodiments, each vascular wall segment was placed onto a porous,
biocompatible membrane within said wells of said container. In some
embodiments, the container is compatible with automated drug
screening. In some embodiments, each vascular wall segment within
the array is affixed to a biocompatible surface on one or more
sides. In further embodiments, the biocompatible surface is a
porous membrane. In still further embodiments, each vascular wall
segment is subjected to shear force, caused by fluid flow, on one
or more sides. In some embodiments, each vascular wall segment
within the array is substantially similar. In other embodiments,
one or more of the vascular wall segments within the array are
unique. In some embodiments, the vascular wall segments within the
array represent one or more distinct vascular tissues in the human
body. In some embodiments, the vascular wall segments within the
array are generated with one or more cell types derived from two or
more distinct human donors. In some embodiments, each vascular wall
segment within the array is maintained independently in culture. In
other embodiments, two or more individual vascular wall segments
within the array exchange soluble factors. In some embodiments, the
array is for use in in vitro assays. In further embodiments, the
array is for use in drug testing. In still further embodiments, the
array is for use in cardiovascular drug testing.
[0187] In certain embodiments, disclosed herein are methods of
constructing a living, three-dimensional vascular wall segment
comprising the steps of: culturing a layer of fibroblasts on a
biocompatible support; preparing a one or more cohered
multicellular aggregates comprising smooth muscle cells, wherein
said aggregates are substantially spherical or substantially
cylindrical; placing one or more cohered multicellular aggregates
onto said support; applying, to said one or more cohered
multicellular aggregates, a layer of endothelial cells on one or
more surfaces; and incubating said multicellular aggregates to
allow them to cohere to form a tissue.
[0188] In certain embodiments, disclosed herein are engineered
tissue culture systems comprising a three-dimensional cell-based
element and a temporary or removable confinement, wherein the
confinement material allows for direct contact between the cells
and a nutrient medium. In some embodiments, the engineered,
three-dimensional cell-based element was bioprinted. In further
embodiments, the engineered, three-dimensional cell-based element
is free of any pre-formed scaffold. In some embodiments, the
confinement material has at least one of the following features:
does not substantially adhere to the cells; is biocompatible; is
extrudable; is non-cellular; is of sufficient strength to provide
support for the cells; and is not soluble in aqueous conditions. In
further embodiments, the confinement material is not plastic, is
not glass, and is not ceramic. In some embodiments, the confinement
material is a hydrogel. In further embodiments, the confinement
material is NovoGel.TM.. In further embodiments, the confinement
material comprises one or more of: agarose, polyethylene glycol
diacrylate (PEG-DA), hyaluronan, gelatin, poloxamer, hydroxyethyl
methacrylate, peptide hydrogel, Matrigel.TM., polydimethylsiloxane,
silicon, silk, polyacrylamide, poly lactic acid, a surfactant
polyol, and alginate. In some embodiments, at least one of: the
cells and/or the confinement material were extruded from a
bioprinter. In further embodiments, there are gaps in the
confinement material and wherein the nutrient medium is capable of
contacting the cells through the gaps. In still further
embodiments, the gaps were between about 100 .mu.m and about 30 mm
wide. In some embodiments, the gaps were distributed non-uniformly
around the structure, whereby the cells of the tissue were exposed
to nutrients non-uniformly. In some embodiments, wherein at least
about 10% of the surface area of the tissue was exposed to gaps
suitable for contact with a nutrient medium. In some embodiments,
the confinement material was overlaid on the cells as at least one
elongated element. In further embodiments, the elongated element of
confinement material had a cross-sectional thickness between about
100 .mu.m and about 1 mm. In some embodiments, there were gaps
between the elongated elements of confinement material. In some
embodiments, gaps were left between elongated elements when
extruding the confinement material from a bioprinter. In other
embodiments, at least some of the confinement material was removed
from the system to provide gaps. In some embodiments, the elongated
elements of confinement material were substantially parallel and
the gaps were elongated. In some embodiments, the elongated
elements of confinement material were arranged in a lattice. In
some embodiments, the elongated elements of confinement material
affix the structure to the supporting surface. In some embodiments,
the system was suitable for shipping. In some embodiments, the
bioprinted cells comprise at least one of: smooth muscle cells,
endothelial cells, fibroblasts, and epithelial cells. In some
embodiments, the nutrient medium comprised at least one of: oxygen
(O.sub.2), a carbon source, a nitrogen source, growth factors,
salts, minerals, vitamins, serum, antibiotics, chemicals, proteins,
nucleic acids, pharmaceutical compounds, and antibodies.
[0189] In certain embodiments, disclosed herein are engineered,
living tissues comprising a three-dimensional cell-comprising
element, held in place by a hydrogel, wherein the hydrogel was
dispensed on said cell-comprising element as cylinders or ribbons
with gaps between the cylinders or ribbons through which the cells
access nutrients, and wherein the hydrogel is removable from the
tissue.
[0190] In certain embodiments, disclosed herein are methods for
increasing the viability of an engineered tissue comprising
providing direct contact between the tissue and a nutrient medium
through a temporary or semi-permanent lattice, wherein the tissue
is free of any pre-formed scaffold. In some embodiments, the step
of providing direct contact between the tissue and a nutrient
medium through a temporary or semi-permanent lattice comprises
constraining said tissue in a porous or gapped material. In further
embodiments, the pores or gaps were between about 100 .mu.m and
about 30 mm wide. In further embodiments, the porous or gapped
material was a hydrogel. In still further embodiments, the porous
or gapped material was agarose. In some embodiments, viability of
an engineered tissue is increased ex vivo. In some embodiments,
viability of at least a portion of the cells comprising an
engineered tissue is extended. In further embodiments, viability of
the cells is extended by 1 day or more. In some embodiments, the at
least one nutrient is selected from the group consisting of: a
carbon source, a nitrogen source, growth factors, salts, minerals,
vitamins, serum, antibiotics, proteins, nucleic acids,
pharmaceutical compounds, ad antibodies. In some embodiments, at
least one nutrient is oxygen (O.sub.2). In further embodiments, the
porous or gapped hydrogel confinement is designed to provide the
bioprinted cells with differential exposure to nutrients on one or
more surfaces.
[0191] In certain embodiments, disclosed herein are methods of
making tissue culture systems comprising the steps of: establishing
a three-dimensional cell-comprising element on a biocompatible
substrate; and dispensing confinement material overlaying the
three-dimensional cell-comprising element, wherein the overlaid
confinement material allows at least some of the cells to contact a
growth medium.
[0192] In certain embodiments, disclosed herein are methods of
making tissue culture systems comprising the steps of: dispensing a
perimeter of confinement material on a surface; dispensing cells
within the perimeter; and dispensing confinement material
overlaying the cells, wherein the overlaid confinement material
allows at least some of the cells to contact a growth medium. In
some embodiments, dispensing confinement material is accomplished
by bioprinting. In some embodiments, the method comprises or
further comprises culturing the system in a suitable medium to
mature the bioprinted cellular construct.
EXAMPLES
[0193] The following specific examples are to be construed as
merely illustrative, and not limitative of the remainder of the
disclosure in any way whatsoever.
Example 1--Cell Culture
Smooth Muscle Cells
[0194] Primary human aortic smooth muscle cells (HASMC;
GIBCO/Invitrogen Corp., Carlsbad, Calif.) were maintained and
expanded in low glucose dulbecco's modified eagle medium (DMEM;
Invitrogen Corp., Carlsbad, Calif.) supplemented with 10% fetal
bovine serum (FBS), 100 U/mL Penicillin, 0.1 mg/mL streptomycin,
0.25 .mu.g/mL of amphotericin B, 0.01M of HEPES (all from
Invitrogen Corp., Carlsbad, Calif.), 50 mg/L of proline, 50 mg/L of
glycine, 20 mg/L of alanine, 50 mg/L of ascorbic acid, and 3
.mu.g/L of CuSO.sub.4 (all from Sigma, St. Louis, Mo.) at
37.degree. C. and 5% CO.sub.2. Confluent cultures of HASMC between
passage 4 and 8 were used in all studies.
Endothelial Cells
[0195] Primary human aortic endothelial cells (HAEC;
GIBCO/Invitrogen Corp., Carlsbad, Calif.) were maintained and
expanded in Medium 199 (Invitrogen Corp., Carlsbad, Calif.)
supplemented with 10% FBS, 1 .mu.g/mL of hydrocortisone, 10 ng/mL
of human epidermal growth factor, 3 ng/mL of basic fibroblast
growth factor, 10 .mu.g/mL of heparin, 100 U/mL Penicillin, 0.1
mg/mL streptomycin, and 0.25 .mu.g/mL of amphotericin B (all from
Invitrogen Corp., Carlsbad, Calif.). The cells were grown on
gelatin (from porcine serum; Sigma, St. Louis, Mo.) coated tissue
culture treated flasks at 37.degree. C. and 5% CO.sub.2. Confluent
cultures of HAEC between passage 4 and 8 were used in all
studies.
Fibroblasts
[0196] Primary human dermal fibroblasts (HDF; GIBCO/Invitrogen
Corp., Carlsbad, Calif.) were maintained and expanded in Medium 106
(Invitrogen Corp., Carlsbad, Calif.) supplemented with 2% FBS, 1
.mu.g/mL of hydrocortisone, 10 ng/mL of human epidermal growth
factor, 3 ng/mL of basic fibroblast growth factor, 10 .mu.g/mL of
heparin, 100 U/mL Penicillin, and 0.1 mg/mL streptomycin (all from
Invitrogen Corp., Carlsbad, Calif.) at 37.degree. C. and 5%
CO.sub.2. Confluent cultures of HDF between passage 4 and 8 were
used in all studies.
SMC-Like Cells from the SVF of Human Lipoaspirate
[0197] SMC-like cells were generated from the adherent fraction of
cells isolated after collagenase digestion of lipoaspirates. This
digestion produces a population of cells known as the stromal
vascular fraction (SVF). The cells of the SVF are plated on
standard tissue culture plastic and adherent cells further selected
with appropriate culture conditions. SMC-like cells from the SVF of
adipose tissue lipoaspirates were maintained and expanded in high
glucose dulbecco's modified eagle medium (DMEM; Invitrogen Corp.,
Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS),
100 U/mL Penicillin, 0.1 mg/mL streptomycin, 0.25 .mu.g/mL of
amphotericin B, 0.01M of HEPES (all from Invitrogen Corp.,
Carlsbad, Calif.), 50 mg/L of proline, 50 mg/L of glycine, 20 mg/L
of alanine, 50 mg/L of ascorbic acid, and 3 .mu.g/L of CuSO.sub.4
(all from Sigma, St. Louis, Mo.) at 37.degree. C. and 5% CO.sub.2.
Confluent subcultures of SVF-SMC between passage 3 and 8 were used
in all studies.
EC from the SVF of Human Lipoaspirate
[0198] Endothelial cells from the stromal vascular fraction (SVF)
were maintained and expanded in growth media that is commonly used
to grow primary isolates of bona fide endothelial cells (EC).
Specifically, SVF-EC were maintained in M199 supplemented with 10%
FBS, 1 .mu.g/mL of hydrocortisone, 10 ng/mL of human epidermal
growth factor, 3 ng/mL basic fibroblast growth factor, 10 .mu.g/mL
of heparin, 100 U/mL Penicillin, and 0.1 mg/mL streptomycin. The
cells were grown on tissue culture-treated flasks at 37.degree. C.
and 5% CO.sub.2. Confluent cultures of SVF-EC between passage 3 and
8 were used in all studies.
Lung-Derived Cells
[0199] Normal Human Lung Fibroblasts were procured from LifeLine
technologies or Lonza and propagated according to manufacturer's
instructions using media from respective vendors. Small Airway
Epithelial Cells were purchased from Lonza and grown in
vendor-provided culture media according to manufacturer's
instructions. Pulmonary airway and pulmonary vascular smooth muscle
cells were obtained from LifeLine Technologies and cultured
according to manufacturer's instructions in vendor-provided
media.
Example 2--NovoGel.TM. Solutions and Mold
[0200] Preparation of 2% and 4% (w/v) NovoGel.TM. Solution
[0201] 1 g or 2 g (for 2% or 4% respectively) of NovoGel.TM.
(Organovo, San Diego, Calif.) was dissolved in 50 mL of Dulbecco's
phosphate buffered saline (DPBS; Invitrogen Corp., Carlsbad,
Calif.). Briefly, the DPBS and NovoGel.TM. are heated to 85.degree.
C. on a hot plate with constant stirring until the NovoGel.TM.
dissolves completely. NovoGel.TM. solution is sterilized by steam
sterilization at 125.degree. C. for 25 minutes. The NovoGel.TM.
solution remains in liquid phase as long as the temperature is
maintained above 65.5.degree. C. Below this temperature a phase
transition occurs, the viscosity of the NovoGel.TM. solution
increases and the NovoGel.TM. forms a solid gel.
Preparation of NovoGel.TM. Mold
[0202] An NovoGel.TM. mold was fabricated for the incubation of
cylindrical bio-ink using a Teflon.RTM. mold that fit a 10 cm Petri
dish. Briefly, the Teflon.RTM. mold was pre-sterilized using 70%
ethanol solution and subjecting the mold to UV light for 45
minutes. The sterilized mold was placed on top of the 10 cm Petri
dish (VWR International LLC, West Chester, Pa.) and securely
attached. This assembly (Teflon.RTM. mold+Petri dish) was
maintained vertically and 45 mL of pre-warmed, sterile 2%
NovoGel.TM. solution was poured in the space between the
Teflon.RTM. mold and the Petri dish. The assembly was then placed
horizontally at room temperature for 1 hour to allow complete
gelation of the NovoGel.TM.. After gelation, the Teflon.RTM. print
was removed and the NovoGel.TM. mold was washed twice using DPBS.
Then 17.5 mL of HASMC culture medium was added to the NovoGel.TM.
mold for incubating the polytypic cylindrical bio-ink.
Example 3--Fabrication of HASMC-HAEC Polytypic Cylindrical
Bio-Ink
[0203] To prepare polytypic cylindrical bio-ink, HASMC and HAEC
were individually collected and then mixed at pre-determined
ratios. Briefly, the culture medium was removed from confluent
culture flasks and the cells were washed with DPBS (1 ml/5 cm.sup.2
of growth area). Cells were detached from the surface of the
culture flasks by incubation in the presence of trypsin (1 ml/15
cm.sup.2 of growth area; Invitrogen Corp., Carlsbad, Calif.) for 10
minutes. HASMC were detached using 0.15% trypsin while HAEC were
detached using 0.1% trypsin. Following the incubation appropriate
culture medium was added to the flasks (2.times. volume with
respect to trypsin volume). The cell suspension was centrifuged at
200 g for 6 minutes followed by complete removal of supernatant
solution. Cell pellets were resuspended in respective culture
medium and counted using a hemocytometer. Appropriate volumes of
HASMC and HAEC were combined to yield a polytypic cell suspension
containing 15% HAEC and remainder 85% HASMC (as a % of total cell
population). The polytypic cell suspension was centrifuged at 200 g
for 5 minutes followed by complete removal of supernatant solution.
Polytypic cell pellets were resuspended in 6 mL of HASMC culture
medium and transferred to 20 mL glass vials (VWR International LLC,
West Chester, Pa.), followed by incubation on an orbital shaker at
150 rpm for 60 minutes, and at 37.degree. C. and 5% CO.sub.2. This
allows the cells to aggregate with one another and initiate
cell-cell adhesions. Post-incubation, the cell suspension was
transferred to a 15 mL centrifuge tube and centrifuged at 200 g for
5 minutes. After removal of the supernatant medium, the cell pellet
was resuspended in 400 .mu.l of HASMC culture medium and pipetted
up and down several times to ensure all cell clusters were broken.
The cell suspension was transferred into a 0.5 mL microfuge tube
(VWR International LLC, West Chester, Pa.) placed inside a 15 mL
centrifuge tube followed by centrifugation at 2000 g for 4 minutes
to form a highly dense and compact cell pellet. The supernatant
medium was aspirated and the cells were transferred into capillary
tubes (OD 1.5 mm, ID 0.5 mm, L 75 mm; Drummond Scientific Co.,
Broomall, Pa.) by aspiration so as to yield cylindrical bio-ink 50
mm in length. The cell paste inside the capillaries was incubated
in HASMC medium for 20 minutes at 37.degree. C. and 5% CO.sub.2.
The cylindrical bio-ink was then extruded from the capillary tubes
into the grooves of a NovoGel.TM. mold (See, e.g., Example 2)
(covered with HASMC medium) using the plunger supplied with the
capillaries. The cylindrical bio-ink was incubated for 24 hours at
37.degree. C. and 5% CO.sub.2.
Example 4--Bioprinting Blood Vessel Wall Segments Comprising HASMC
and HAEC Polytypic Cylindrical Bio-Ink
[0204] Blood vessel wall-mimicking segments were bioprinted
utilizing a NovoGen MMX Bioprinter.TM. (Organovo, Inc., San Diego,
Calif.) either on NovoGel.TM. base plates (100 mm Petri dish size),
inside NovoGel.TM. coated wells, or directly onto Corning.RTM.
Transwell.RTM. inserts in a multi-well plate (e.g., 6-well plates).
This process involves the following three phases:
Preparation of HASMC-HAEC Polytypic Bio-Ink
[0205] Cultures of human aortic smooth muscle cells (HASMC) and
human aortic endothelial cells (HAEC) were trypsinized, counted,
and mixed in appropriate quantities to yield bio-ink that comprised
HASMC:HAEC at either a 85:15 or 70:30 ratio. The polytypic cell
suspension was shaken for 60 minutes on a rotary shaker, collected,
and centrifuged. Cells were drawn into 266 or 500 .mu.m (ID) glass
microcapillaries, then extruded into media covered NovoGel.TM.
plates and incubated for a minimum of 6 hours.
Bioprinting of Patches/Three-Dimensional Cell Sheets
[0206] In the case of printing onto NovoGel.TM. beds inside the
wells of a multi-well plate or on NovoGel.TM. base plates (100 mm
Petri dish size), a first layer of NovoGel.TM. cylinders was
bioprinted. Then, on top of it a box was bioprinted using
NovoGel.TM. rods such that the space inside was 8 mm
long.times.1.25 mm wide. Matured cylindrical bio-ink was loaded
onto the bioprinter for printing inside the box. Finally, a third
layer of NovoGel.TM. cylinders was printed on top of the second
that either covers the entire length of cells or creates a
lattice/mesh type structure on top. In the case of printing onto
Transwell.RTM. inserts inside the wells of the plate, the first
layer of NovoGel.TM. rods described earlier was eliminated. The
bioprinted constructs were then covered with appropriate cell
culture medium and incubated during which the adjoining segments of
the bio-ink fused to form a three-dimensional patch of cells.
Maturation of Bioprinted Constructs
[0207] The bioprinted constructs comprising the HASMC-HAEC bio-ink
were incubated for a period of 1-7 days to allow the construct to
mature and provide the HAEC sufficient time to sort to the
periphery of the construct thereby mimicking a section of a blood
vessel wall. In some experiments, the three-dimensional cellular
patch was subjected to shear forces (i.e., pulsatile flow) to aid
the process of HAEC sorting.
Example 5--Fabrication of HASMC-HDF-HAEC Polytypic Cylindrical
Bio-Ink
[0208] To prepare polytypic cylindrical bio-ink, HASMC, HDF, and
HAEC were individually collected and then mixed at pre-determined
ratios (e.g., HASMC:HDF:HAEC ratios of 70:25:5). Briefly, the
culture medium was removed from confluent culture flasks and the
cells were washed with DPBS (1 ml/10 cm2 of growth area). Cells
were detached from the surface of the culture flasks by incubation
in the presence of trypsin (1 ml/15 cm2 of growth area; Invitrogen
Corp., Carlsbad, Calif.) for 10 minutes. HASMC and HDF were
detached using 0.15% trypsin while HAEC were detached using 0.1%
trypsin. Following the incubation appropriate culture medium was
added to the flasks (2.times. volume with respect to trypsin
volume). The cell suspension was centrifuged at 200 g for 6 minutes
followed by complete removal of supernatant solution. Cell pellets
were resuspended in respective culture medium and counted using a
hemocytometer. Appropriate volumes of HASMC, HDF, and HAEC were
combined to yield polytypic cell suspensions. The polytypic cell
suspensions were centrifuged at 200 g for 5 minutes followed by
aspiration of the supernatant solution. Polytypic cell pellets were
resuspended in 6 mL of HASMC culture medium and transferred to 20
mL glass vials (VWR International LLC, West Chester, Pa.), followed
by incubation on an orbital shaker at 150 rpm for 60 minutes, and
at 37.degree. C. and 5% CO.sub.2. This allows the cells to
aggregate with one another and initiate cell-cell adhesions.
Post-incubation, the cell suspension was transferred to a 15 mL
centrifuge tube and centrifuged at 200 g for 5 minutes. After
removal of the supernatant medium, the cell pellet was resuspended
in 400 .mu.L of HASMC culture medium and pipetted up and down
several times to ensure all cell clusters were broken. The cell
suspension was transferred into a 0.5 mL microfuge tube (VWR
International LLC, West Chester, Pa.) placed inside a 15 mL
centrifuge tube followed by centrifugation at 2000 g for 4 minutes
to form a highly dense and compact cell pellet. The supernatant
medium was aspirated and the cells were transferred into capillary
tubes (OD 1.25 mm, ID 0.266 mm, L 75 mm; Drummond Scientific Co.,
Broomall, Pa.) by aspiration so as to yield cylindrical bio-ink
aggregates 50 mm in length. The cell paste inside the capillaries
was incubated in HASMC medium for 20 minutes at 37.degree. C. and
5% CO.sub.2. The cylindrical bio-ink was then extruded from the
capillary tubes into the grooves of a NovoGel.TM. mold (see, e.g.,
Example 2) (covered with HASMC medium) using the plunger supplied
with the capillaries. The cylindrical bio-ink was incubated for 6
to 24 hours at 37.degree. C. and 5% CO.sub.2.
Example 6--Bioprinting Blood Vessel Wall Segments Comprising
Polytypic HASMC, HAEC, and HDFa Bio-Ink
[0209] Blood vessel wall-mimicking segments were bioprinted
utilizing a NovoGen MMX Bioprinter.TM. (Organovo, Inc., San Diego,
Calif.) either on NovoGel.TM. base plates (100 mm Petri dish size),
inside NovoGel.TM. coated wells, or directly onto Corning.RTM.
Transwell.RTM. inserts in a multi-well plate (e.g., 6-well plates).
This process involves the following three phases:
Preparation of Polytypic HASMC-HDFa-HAEC Bio-Ink
[0210] Cultures of HASMC, HAEC, and HDFa were trypsinized, counted,
and mixed in appropriate quantities to yield bio-ink that comprised
HASMC:HDFa:HAEC at a 70:15:15 ratio. The polytypic cell suspension
was shaken for 60 minutes on a rotary shaker, collected, and
centrifuged. Cells were drawn into 266 or 500 .mu.m (ID) glass
microcapillaries, then extruded into media covered NovoGel.TM.
plates and incubated for a minimum of 6 hours.
Bioprinting of Patches/Three-Dimensional Cell Sheets
[0211] In the case of printing onto NovoGel.TM. beds inside the
wells of a multi-well plate or on NovoGel.TM. base plates (100 mm
Petri dish size), a first layer of NovoGel.TM. cylinders was
bioprinted. Then, on top of it a box was bioprinted using
NovoGel.TM. rods such that the space inside was 8 mm
long.times.1.25 mm wide. Matured cylindrical bio-ink aggregates
were loaded onto the bioprinter for printing inside the box.
Finally, a third layer of NovoGel.TM. cylinders was printed on top
of the second that either covers the entire length of cells or
creates a lattice/mesh type structure on top. In the case of
printing onto Transwell.RTM. inserts inside the wells of the plate,
the first layer of NovoGel.TM. rods described earlier was
eliminated. The bioprinted constructs were then covered with
appropriate cell culture medium and incubated during which the
adjoining segments of the bio-ink fused to form a three-dimensional
patch of cells.
Maturation of Bioprinted Constructs
[0212] The bioprinted constructs comprising polytypic
HASMC-HDFa-HAEC bio-ink were incubated for a period of 1-7 days to
allow the construct to mature and provide the HAEC sufficient time
to sort to the periphery of the construct thereby mimicking a
section of a blood vessel wall. In some experiments, the
three-dimensional cellular patch was subjected to shear forces
(i.e., pulsatile flow) to aid the process of HAEC sorting.
Example 7--Fabrication of SVF-SMC-SVF-EC Polytypic Cylindrical
Bio-Ink
[0213] To prepare polytypic cylindrical bio-ink, SVF-SMC and SVF-EC
were individually collected and then mixed at pre-determined
ratios. Briefly, the culture medium was removed from confluent
culture flasks and the cells were washed with DPBS (1 ml/5 cm.sup.2
of growth area). Cells were detached from the surface of the
culture flasks by incubation in the presence of TrypLE (Invitrogen
Corp., Carlsbad, Calif.) for 5 to 10 minutes. Following the
incubation appropriate culture medium was added to the flasks to
quench enzyme activity. The cell suspension was centrifuged at 200
g for 6 minutes followed by complete removal of supernatant
solution. Cell pellets were resuspended in respective culture
medium and counted using a hemocytometer. Appropriate volumes of
SVF-SMC and SVF-EC were combined to yield a polytypic cell
suspension containing 15% SVF-EC and remainder 85% SVF-SMC (as a %
of total cell population). The polytypic cell suspension was
centrifuged at 200 g for 5 minutes followed by complete removal of
supernatant solution. Polytypic cell pellets were resuspended in 6
mL of SVF-SMC culture medium and transferred to 20 mL glass vials
(VWR International LLC, West Chester, Pa.), followed by incubation
on an orbital shaker at 150 rpm for 60 minutes, and at 37.degree.
C. and 5% CO.sub.2. This allows the cells to aggregate with one
another and initiate cell-cell adhesions. Post-incubation, the cell
suspension was transferred to a 15 mL centrifuge tube and
centrifuged at 200 g for 5 minutes. After removal of the
supernatant medium, the cell pellet was resuspended in 400 .mu.l of
SVF-SMC culture medium and pipetted up and down several times to
ensure all cell clusters were broken. The cell suspension was
transferred into a 0.5 mL microfuge tube (VWR International LLC,
West Chester, Pa.) placed inside a 15 mL centrifuge tube followed
by centrifugation at 2000 g for 4 minutes to form a highly dense
and compact cell pellet. The supernatant medium was aspirated and
the cells were transferred into capillary tubes (OD 1.25 mm, ID
0.266 mm, L 75 mm; Drummond Scientific Co., Broomall, Pa.) by
aspiration so as to yield cylindrical bio-ink aggregates 50 mm in
length. The cell paste inside the capillaries was incubated in
SVF-SMC medium for 20 minutes at 37.degree. C. and 5% CO.sub.2. The
cylindrical bio-ink was then extruded from the capillary tubes into
the grooves of a NovoGel.TM. mold (see, e.g., Example 2) (covered
with SVF-SMC medium) using the plunger supplied with the
capillaries. The cylindrical bio-ink was incubated for 6 to 12
hours at 37.degree. C. and 5% CO.sub.2.
Example 8--Bioprinting Blood Vessel Wall Segments Comprising a
Mixture of Vascular SMC and EC
[0214] Blood vessel wall constructs were bioprinted utilizing a
NovoGen MMX Bioprinter.TM. (Organovo, Inc., San Diego, Calif.) into
the wells of 6-well culture plates that had been previously covered
with 1.5 mL of 2% (w/v) NovoGel.TM.. Cylindrical bio-ink was
prepared with a mixture of human vascular smooth muscle cells (SMC)
and human endothelial cells (EC) at an SMC:EC ratio of 85:15 or
70:30. Bio-ink was generated by aspiration of a cell pellet
(SMC:EC) into a glass microcapillary tube with either a 500 .mu.m
or 266 .mu.m inner diameter (ID). The bio-ink cylinders were then
extruded into a NovoGel.TM. mold covered with appropriate culture
medium. Prior to bioprinting, the bio-ink was held for 6 to 18
hours. Polytypic bio-ink containing a mixture of SMC and EC was
used. In these experiments the EC within the bio-ink sorted to the
periphery of the bio-ink aggregates, resulting in a construct that
is covered with EC and contains a SMC-rich construct wall. This
process resulted in the development of a vascular wall construct
that contains a wall comprised of SMC and a covering of EC (e.g., a
putative tunica media and tunica intima). The constructs were
bioprinted in the center of the culture well using bioprinting
protocols and the culture well was filled with appropriate culture
media and the constructs returned to the incubator for maturation
and evaluation. Following bioprinting, the construct was covered
with an appropriate amount of culture media (e.g., 4 mL for 1 well
of a 6-well plate). In summary, this example describes the use of
vascular SMC and EC for bioprinting a vascular wall segment or
mimic within a standard size multi-well tissue culture plate. The
resulting vessel wall segment or mimic is characterized by an
external layer or layers of EC and internal wall comprised largely
or solely of SMC.
Example 9--Bioprinting Blood Vessel Wall Segments Comprising Human
Vascular SMC with a Covering of EC
[0215] Blood vessel wall constructs were bioprinted utilizing a
NovoGen MMX Bioprinter.TM. (Organovo, Inc., San Diego, Calif.) into
the wells of 6-well culture plates that had been previously covered
with 1.5 mL of 2% (w/v) NovoGel.TM.. Cellular bio-ink was prepared
with human vascular smooth muscle cells (SMC). Bio-ink cylinders
were generated by aspiration of a cell pellet (SMC) into a glass
microcapillary tube with either a 500 .mu.m or 266 .mu.m inner
diameter (ID). The cylindrical bio-ink aggregates were then
extruded into a NovoGel.TM. mold covered with appropriate culture
medium. Prior to bioprinting, the bio-ink was held for 6 to 18
hours. An EC-concentrate (1-1.5.times.10.sup.5 cells/.mu.l) was
bioprinted directly on top of the previously bioprinted SMC
structure to form a second layer of the construct. This process
resulted in the development of a vascular wall construct that
contains a wall comprised of SMC and a covering of EC (e.g., a
putative tunica media and tunica intima). The constructs were
bioprinted in the center of the culture well using bioprinting
protocols. Following bioprinting, the construct was covered with an
appropriate amount of culture media (e.g., 4 mL for 1 well of a
6-well plate) and returned to the incubator for maturation and
evaluation. In summary, this example describes the use of vascular
SMC and EC for bioprinting a vascular wall segment or mimic within
a standard size multi-well tissue culture plate. The resulting
vessel wall segment or mimic is characterized by an external layer
of EC and internal wall comprised largely or solely of SMC.
Example 10--Bioprinting Blood Vessel Wall Segments Comprising HASMC
Layered with HAEC Utilizing NovoGel.TM. Containment
[0216] Blood vessel wall-mimicking segments were bioprinted
utilizing a NovoGen MMX Bioprinter.TM. (Organovo, Inc., San Diego,
Calif.) either inside NovoGel.TM. coated wells or directly onto
Corning.RTM. Transwell.RTM. inserts in a multi-well plate (e.g.,
6-well plates). This process involved the following three
phases:
Preparation of HASMC Bio-Ink
[0217] Cultures of human aortic smooth muscle cells (HASMC) were
trypsinized, and then shaken for 60 minutes on a rotary shaker.
Post-shaking, cells were collected, centrifuged, and aspirated into
266 or 500 .mu.m (ID) glass microcapillaries. Finally, the cells
were extruded into media covered NovoGel.TM. plates and incubated
for a minimum of 6 hours.
Bioprinting of HASMC Patches Layered with HAEC
[0218] Just prior to bioprinting of patches (e.g., segments), human
aortic endothelial cell (HAEC) cultures were trypsinized, counted,
and then resuspended in HAEC medium at a working concentration of
1.times.10.sup.6 cells/10 .mu.L of medium. The HAEC suspension was
placed in the bioprinter to be utilized for layering bioprinted
patches. In the case of printing onto NovoGel.TM. beds inside the
wells of a multi-well plate, a first layer of NovoGel.TM. cylinders
was bioprinted. Then, on top of it a box was bioprinted using
NovoGel.TM. rods such that the space inside was 8 mm
long.times.1.25 mm wide. Matured cylindrical HASMC bio-ink was
loaded onto the bioprinter for printing inside the box. HAEC in
suspension were then drawn into a clean microcapillary by the
bioprinter and dispensed on top of the printed HASMC layer 4 times
near the 4 corners of the printed patch. Each drop was 2.5 .mu.L in
volume. The construct was incubated for a period of 15-30 minutes
before proceeding to print the third layer. Finally, a third layer
of NovoGel.TM. cylinders was printed on top of the second to create
a lattice/mesh type structure on top. In the case of printing onto
Transwell.RTM. inserts inside the wells of the plate, the first
layer of NovoGel.TM. rods described earlier was eliminated. The
bioprinted constructs were then covered with appropriate cell
culture medium and incubated.
Maturation of Bioprinted Constructs
[0219] The bioprinted constructs were incubated for a period of 1-7
days to allow the construct to mature and provide the HAEC
sufficient time to form a uniformly thin monolayer on top of the
HASMC patch. In some experiments, the three-dimensional cellular
patch was subjected to shear forces (i.e., pulsatile flow).
Example 11--Bioprinting Blood Vessel Wall Segments Comprising HASMC
Layered with HAEC onto a HDFa Monolayer Utilizing NovoGel.TM.
Containment
[0220] Blood vessel wall-mimicking segments were bioprinted
utilizing a NovoGen MMX Bioprinter.TM. (Organovo, Inc., San Diego,
Calif.) directly onto Corning.RTM. Transwell.RTM. inserts in a
multi-well plate (e.g., 6-well plates). This process involved the
following four phases:
Culture of HDFa's onto Transwelly Membranes
[0221] Human adult dermal fibroblasts (HDFa) were seeded onto
Transwell.RTM. membranes at a density of 20,000 cells/cm.sup.2 and
cultured for a minimum of 6 days. This allowed the cells to adhere,
grow and become a confluent layer on the Transwell.RTM.
membrane.
Preparation of HASMC Bio-Ink
[0222] Cultures of human aortic smooth muscle cells (HASMC) were
trypsinized, and shaken for 60 minutes on a rotary shaker.
Post-shaking, cells were collected, centrifuged, and aspirated into
266 or 500 .mu.m (ID) glass microcapillaries. The cells were then
extruded into media covered NovoGel.TM. plates and incubated for a
minimum of 6 hours.
Bioprinting of HASMC Patches Layered with HAEC
[0223] Just prior to bioprinting of patches (e.g., segments), human
aortic endothelial cell (HAEC) cultures were trypsinized, counted,
and then resuspended in HAEC medium at a working concentration of
1.times.10.sup.6 cells/10 .mu.L of medium. The HAEC suspension was
placed in the bioprinter to be utilized for layering bioprinted
patches. The culture media in the multi-well plates having the
HDFa's grown on Transwell.RTM. membranes was completely aspirated
and the plate transferred to the bioprinter. A box was bioprinted
using NovoGel.TM. rods such that the space defined was 8 mm
long.times.1.25 mm wide directly on top of the HDFa's on the
membrane. Matured HASMC bio-ink cylinders were loaded onto the
bioprinter for printing inside the box. HAEC in suspension were
then drawn into a clean microcapillary tube by the bioprinter and
dispensed on top of the printed HASMC layer 4 times near the 4
corners of the printed patch. Each drop was 2.5 .mu.L in volume.
The construct was incubated for a period of 15-30 minutes before
proceeding to print the top NovoGel.TM. rod layer. Finally, a top
layer of NovoGel.TM. cylinders was printed to create a lattice/mesh
type structure. The bioprinted constructs were then covered with
appropriate cell culture medium and incubated.
Maturation of Bioprinted Constructs
[0224] The bioprinted constructs were incubated for a period of 1-7
days to allow the construct to mature and provide the HAEC
sufficient time to form a uniformly thin monolayer on top of the
HASMC patch.
Example 12--Hydrogel Lattice Used to Spatially Confine a Construct
while Allowing for Direct Contact with Media
[0225] Cylindrical hydrogel elements were bioprinted utilizing a
NovoGen MMX Bioprinter.TM. (Organovo, Inc., San Diego, Calif.)
across a portion of the top surface of a three-dimensional cell
sheet. The lattice provided spatial confinement to the sheet and
allowed for direct contact between the sheet and the surrounding
media. First, a hydrogel base layer was bioprinted. Second, a
hydrogel window was bioprinted defining a space 8 mm
long.times.1.25 mm wide. Third, cellular bio-ink was bioprinted
inside the hydrogel window to form the three-dimensional cell
sheet. And, fourth, the hydrogel lattice structure was bioprinted.
In various experiments, the size of the hydrogel elements was
approximately 100 .mu.m to 1 mm in diameter, and the spacing
between the elements was approximately 100 .mu.m to 10 mm.
[0226] In some experiments, the hydrogel elements were printed
along one direction to create long open channels on top of the cell
sheet. In other experiments, the hydrogel elements were printed in
multiple directions to create a grid-like pattern of open areas on
top of the sheet. The hydrogel was comprised of NovoGel.TM.. The
lattice structure was optionally extended past the structure and
onto the print surface to allow for the application of additional
material to affix the structure to the print surface. The resulting
lattice was used to spatially confine the construct, but allow for
some of the cellular construct to have direct contact with the
surrounding nutritive media.
Example 13--Liver Tissue Bioprinted Using Continuous Deposition and
Tessellated Functional Unit Structure
[0227] Engineered liver tissue was bioprinted utilizing a NovoGen
MMX Bioprinter.TM. (Organovo, Inc., San Diego, Calif.) using a
continuous deposition mechanism. The three-dimensional structure of
the liver tissue was based on a repeating functional unit, in this
case, a hexagon. The bio-ink was composed of hepatic stellate cells
and endothelial cells encapsulated in an extrusion compound
(surfactant polyol--PF-127).
Preparation of 30% PF-127
[0228] A 30% PF-127 solution (w/w) was made using PBS. PF-127
powder was mixed with chilled PBS using a magnetic stir plate
maintained at 4.degree. C. Complete dissolution occurred in
approximately 48 hours.
Cell Preparation and Bioprinting
[0229] A cell suspension comprised of 82% stellate cells (SC) and
18% human aortic endothelial cells (HAEC) and human adult dermal
fibroblasts (HDFa) was separated into 15 mL tubes in order to
achieve three cell concentrations: 50.times.10.sup.6 cells/ml,
100.times.10.sup.6 cells/ml, and 200.times.10.sup.6 cells/mL
following centrifugation. Each cell pellet was resuspended in 30%
PF-127 and aspirated into a 3 cc reservoir using the bioprinter.
With a 510 .mu.m dispense tip, the encapsulated cells were
bioprinted onto a PDMS base plate into a single hexagon (see FIG.
6A) or hexagon tessellation configuration (see FIG. 6B). Each
construct received approximately 200 .mu.L of media and was
incubated for 20 minutes at room temperature to evaluate construct
integrity.
Multi-Layer Bioprinting
[0230] For hexagon tessellation experiments, up to (4) sequential
layers were bioprinted resulting in a taller structure with more
cellular material present. Following fabrication, each construct
initially received approximately 200 .mu.L of complete media to
assess construct integrity. Constructs were incubated for 20
minutes at room temperature and were then submerged in 20 mLs of
complete media.
Results
[0231] Following 18 hours of culture in growth media containing 10%
fetal bovine serum (which dissolves PF127), cells contained within
the bioprinted geometry were cohered to each other sufficiently to
generate an intact, contiguous sheet of tissue that retained the
geometrical patterning of the original design (see FIG. 6D). Shown
in FIG. 7 is H&E staining of a single segment of the
tessellated construct, after fixation in 10% neutral buffered
formalin. Cells were found to be viable, intact, and confined to
their original printed geometry.
Example 14--Forced Layering
[0232] Cell populations (homogeneous or heterogeneous) were
prepared for bioprinting as either cylindrical bio-ink or as a cell
suspension in Pluronic F-127 (Lutrol, BASF). Briefly, for
preparation of bio-ink, cells were liberated from standard tissue
culture plastic using either recombinant human trypsin (75
.mu.g/mL, Roche) or 0.05% trypsin (Invitrogen). Following enzyme
liberation, cells were washed, collected, counted and combined at
desired ratios (i.e., 50:50 hepatic stellate cell (hSC):endothelial
cell (EC)) and pelleted by centrifugation. Supernatant was then
removed from the cell pellet and the cell mixture was aspirated
into a glass microcapillary of desired diameter, typically 500
.mu.m or 250 .mu.m, internal diameter. This cylindrical cell
preparation was then extruded into a mold, generated from non
cell-adherent hydrogel material with channels for bio-ink
maturation. The resulting bio-ink cylinders were then cultured in
complete cell culture media for an empirically determined amount of
time, typically 2 to 24 hours.
[0233] Briefly, for hydrogel cell suspension preparation, cells
were liberated from standard cell culture vessel using either of
the enzyme-mediated protocols described herein. Liberated cells
were then washed with serum containing media, collected, counted
and centrifuged to form a dense cell pellet. Supernatant was
removed from the resulting cell pellet and cells were then
resuspended in cold PF-127 (4.degree. C.) at a concentration of 50
to 200.times.10.sup.6 cells/mL (ranging from 10 to
300.times.10.sup.6 cells/mL). This cell suspension was then
aspirated into a syringe, utilizing a NovoGen MMX Bioprinter.TM.
(Organovo, Inc., San Diego, Calif.).
[0234] Fabrication of tissue constructs with forced cell
patterning, layering, or orientation was then accomplished using
the bioprinter. Bioprinting of three-dimensional tissue constructs
was performed with cylindrical bio-ink, cellular suspensions in
water soluble gels, or combinations thereof. To achieve defined
cell patterning or layering, combinations of relevant cell
populations were included in the bio-ink or cell suspension
preparation and then bioprinted in such a fashion that dissolution
of the gel material supporting the cell solution, results in
defined cell layering around the deposited bio-ink (see FIG. 8).
Cell patterning, organization, or layering was also achieved
through the utilization and incorporation of defined, discrete cell
populations (e.g., hSC and EC), which resulted in predictable and
repeatable organization of cells and cellular structures within the
bioprinted tissues (see FIG. 9).
[0235] In some experiments, final cellular organization within the
bioprinted neotissue was observed after a maturation or culture
period. Constructs were maintained in a standard laboratory
incubator (37.degree. C., humidified chamber supplemented with 5%
CO.sub.2) and evaluated over time.
Results
[0236] Cell patterning, layering, or arrangement was achieved using
bioprinting. By bioprinting with bio-ink containing heterogeneous
(i.e., polytypic) cell populations, or by combining bio-ink
(homogeneous or heterogeneous cell populations) with high density
cell-gel or cell suspensions, distinct cell organization was
observed. Maturation of these neotissue constructs in a humidified
chamber (incubator) resulted in further establishment of distinct
cell arrangement, organization and/or segregation in these
bioprinted neotissues.
[0237] For example, bioprinting of EC:hSC-laden PF-127 on top of
bioprinted bio-ink comprising HepG2 cells results in the
establishment of distinct layers of the construct with distinct
cell populations and discreet tissue morphology. In the case of
bio-ink constructs containing hSC and EC, bioprinted constructs
that were matured for more than 3 days in complete media were found
to contain a distinct layer of EC at the periphery and organized
microvessel networks within the core of the construct. Bioprinted
constructs fabricated with bio-ink comprising a homogeneous (i.e.,
monotypic) population of vascular smooth muscle cell onto which a
highly concentrated solution of EC was bioprinted were found to
contain a distinct layer of EC at the periphery of the
construct.
Example 15--Layered Non-Blood Vessel Constructs (Airway
Analogues)
[0238] Cylindrical bio-ink was prepared with normal human lung
fibroblasts (NHLF), small airway epithelial cells (SAEC) and human
aortic endothelial cells (EC). Cells were propagated under standard
laboratory conditions and cells were cultured in media either
purchased from the same vendor as the cells, or media comprising
components typically found in the primary literature to be
conducive to standard cell culture practices for those particular
cell types. Briefly, cells were liberated from standard tissue
culture plastic by washing with cation-free phosphate buffered
saline (PBS) and then exposed to 0.1-0.05% trypsin (Invitrogen).
Liberated cells were then washed in serum-containing media,
collected, counted and combined in an appropriate ratio and
pelleted by centrifugation. Typically, NHLF and EC were mixed in a
ratio of 90:10 to 50:50, NHLF:EC. Supernatant was then removed and
the cell pellet was aspirated into a glass microcapillary, which
was then submerged in complete media for approximately 15 to 20
minutes. This cylindrical bio-ink structure was then extruded into
a non cell-adherent hydrogel mold, containing linear channels and
held for 2 to 18 hours.
[0239] SAEC were then prepared in a highly concentrated cell
suspension. Briefly, SAEC were liberated as described herein,
collected, enumerated, and pelleted by centrifugation. Supernatant
was removed and the cell pellet was resuspended in a small volume
of complete media, yielding a highly concentrated cell pellet of
1.times.10.sup.5 cells/.mu.L. This cell suspension was then stored
at 4.degree. C. until time of use.
[0240] Human lung constructs were then bioprinted into the wells of
a multi-well plate or onto the membrane of a cell culture well
insert (Transwell, BD). Multicellular NHLF or NHLF:EC bio-ink was
used to bioprint a layer of tissue representing the small airway
wall. Human airway tissue segments were fabricated with initial
dimensions of 1.25 mm.times.8 mm.times.0.25 mm (W.times.L.times.H).
Following bioprinting of the wall layer with NHLF or NHLF:EC
bio-ink, a concentrated cell suspension of SAEC was bioprinted on
the top surface of the wall, generating a second layer comprising
airway epithelium on top of putative airway interstitium (see FIG.
10).
[0241] The human airway tissue segments were then submerged in
serum-containing complete cell culture media and placed in a
standard humidified chamber, supplemented with 5% CO.sub.2 for
maturation. The bioprinted human airway segments were then cultured
in static conditions or stimulated through the addition of
cytokine(s) or biomechanical signals (e.g., flow, shear stress,
etc.). Bioprinted human lung tissue constructs were then cultured
for up to 7 days and evaluated for cell organization, extracellular
matrix production, cell viability, and construct integrity (see
FIG. 11).
Results
[0242] Bioprinted human lung tissue constructs with a layered
cellular structure comprising an NHLF wall containing an organized
network of EC-lined microvessel profiles and an apical surface
comprising small airway epithelial cells were successfully
fabricated and maintained in culture. The bioprinted constructs
were generated using a multi-layered approach with NHLF or NHLF:EC
bio-ink cylinders and a bioprinted layer of SAEC. Upon stimulation
with a cytokine believed to be important in disease processes of
the lung, morphological changes including tissue thickening and
NHLF activation were observed.
Example 16--Layered Blood Vessel Wall Constructs
[0243] Cylindrical bio-ink was prepared with vascular smooth muscle
cells (SMC) and, in some experiments, dermal fibroblasts (Fb).
Briefly, cells were liberated from standard tissue culture plastic
by washing with cation-free phosphate buffered saline (PBS) and
then exposed to 0.05% trypsin (Invitrogen). Liberated cells were
then washed in serum-containing media, collected, counted and, for
experiments in which Fb were included, combined in an appropriate
ratio and pelleted by centrifugation. Supernatant was then removed
and the cell pellet was aspirated into a glass microcapillary,
which was then submerged in complete media for approximately 15 to
20 minutes. This cylindrical bio-ink structure was then extruded
into a non cell-adherent hydrogel mold, containing linear channels
and held for 2 to 18 hours.
[0244] Endothelial cells (EC) were then prepared in a highly
concentrated cell suspension. Briefly, EC were liberated as
described above, collected, enumerated, and pelleted by
centrifugation. Supernatant was removed and the cell pellet was
resuspended in a small volume of complete media, yielding a highly
concentrated cell pellet of 1.times.10.sup.5 cells/.mu.L. This cell
suspension was then stored at 4.degree. C. until time of use.
[0245] Blood vessel wall constructs were then bioprinted into the
wells of a multi-well plate or onto the membrane of a cell culture
well insert (Transwell, BD). Cylindrical SMC or SMC:Fb bio-ink was
used to bioprint the tunica media of a blood vessel wall segment.
Blood vessel wall segments were fabricated with initial dimensions
of 1.25 mm.times.8 mm.times.0.25 mm (W.times.L.times.H). Following
bioprinting of the putative tunica media with SMC or SMC:Fb bio-ink
to form a first layer of tissue, a concentrated cell suspension of
EC was bioprinted on the top surface of the first layer to generate
a second layer of vascular endothelium, serving as a putative
tunica intima (see FIG. 12).
[0246] The bioprinted blood vessel wall segments were then
submerged in serum-containing complete cell culture media and
placed in a standard humidified chamber, supplemented with 5%
CO.sub.2 for maturation. The bioprinted blood vessel wall segments
were then cultured in static conditions or stimulated through the
addition of cytokine(s) or biomechanical signals (e.g., flow, shear
stress, etc.). Blood vessel wall segments were cultured for up to 7
days and evaluated for cell organization, extracellular matrix
production, cell viability and construct integrity (see FIG.
13).
Results
[0247] Bioprinted vascular wall segments with a layered cellular
structure comprising an SMC-rich media and an EC-lined intima were
successfully fabricated and maintained in culture. The bioprinted
constructs were generated using a multi-layered approach with SMC
or SMC:Fb bio-ink cylinders and a bioprinted layer of EC.
Example 17--Multi-Well Plates
[0248] Cell populations (homogeneous or heterogeneous) were
prepared for bioprinting using a variety of bio-ink formats,
including cylindrical bio-ink aggregates, suspensions of cellular
aggregates, or cell suspensions/pastes, optionally containing
extrusion compounds. Briefly, for preparation of cylindrical
bio-ink, cells were liberated from standard tissue culture plastic
using either recombinant human trypsin (75 .mu.g/mL, Roche) or
0.05% trypsin (Invitrogen). Following enzyme liberation, cells were
washed, collected, counted, and combined at desired ratios (i.e.,
50:50 hepatic stellate cell (hSC):endothelial cell (EC)) and
pelleted by centrifugation. Supernatant was then removed from the
cell pellet and the cell mixture was aspirated into a glass
microcapillary of desired diameter, typically 500 .mu.m or 250
.mu.m, internal diameter. This cylindrical cell preparation was
then extruded into a mold, generated from non cell-adherent
hydrogel material with channels for bio-ink maturation. The
resulting bio-ink cylinders were then cultured in complete cell
culture media for an empirically determined amount of time,
typically 2 to 24 hours.
[0249] For preparation of a cell suspension or cell paste of
cellular aggregates, the cell propagation and liberation protocols
described herein were followed. At the time of cell pellet
generation, supernatant was removed from the pellet and the pellet
was transferred to a custom deposition syringe. This syringe was
then mounted to the bioprinter deposition head for direct
bioprinting of the cell aggregate solution or paste into multi-well
plates.
[0250] Replicate tissue constructs were then bioprinted within the
wells of either a multi-well tissue culture plate (e.g., 6-well or
24-well) or within a multi-well cell culture insert (i.e.,
Transwell, BD) and then placed into an appropriate multi-well
plate. Following bioprinting, the three-dimensional constructs were
matured/conditioned with relevant media for some period of time,
typically 3 to 14 days. Following maturation, constructs were
harvested, fixed and processed for routine histology and
immunohistochemistry.
Results
[0251] Bioprinted tissues were successfully fabricated within
multi-well culture plates or multi-well culture inserts that were
then inserted into multi-well plates. This approach allows for
generation of replicate bioprinted tissues that are optionally
cultured and treated to generate identical or unique culture
conditions. This approach results in a significant increase in
bioprinting process throughput and sample generation (see FIG.
14).
Example 18--Stimulation of Bioprinted Neotissues
[0252] Cylindrical bio-ink comprising relevant heterogeneous (i.e.,
polytypic) cell populations were prepared. Physiologically-relevant
populations (e.g., normal human lung fibroblasts (NHLF) and small
airway epithelial cells (SAEC) or vascular smooth muscle cells
(SMC) and vascular endothelial cells (EC)) of cells were combined
at specific ratios to generate proper bio-ink. In additional
experiments, hepatic stellate cells (hSCs) were combined with ECs
to generate liver tissue. In additional experiments, hepatic
stellate cells (hSCs) were combined with ECs to generate liver
tissue. Cells were maintained and propagated under standard
laboratory conditions and cells were cultured in media either
purchased from the same vendor as the cells, or media comprising
components typically found in the primary literature to be
conducive to standard cell culture practices for those particular
cell types. Cell processing for bio-ink preparation was as follows:
briefly, cells were liberated from standard tissue culture plastic
by washing with cation-free phosphate buffered saline (PBS) and
then exposed to 0.1-0.05% trypsin (Invitrogen). Liberated cells
were then washed in serum-containing media, collected, counted, and
combined in an appropriate ratio for the stimulation assay or
experiment being conducted, and pelleted by centrifugation.
Supernatant was then removed and the cell pellet was aspirated into
a glass microcapillary, which was then submerged in complete media
for approximately 15 to 20 minutes. This cylindrical bio-ink
structure was then extruded into a non cell-adherent hydrogel mold,
containing linear channels and held for 2 to 18 hours.
[0253] For tissue constructs requiring a homogeneous (i.e.,
monotypic) cell layer, restricted to the upper surface, a secondary
cell preparation was utilized containing the relevant cell type.
Typically vascular endothelial cells or small airway epithelial
cells (for blood vessel wall and human lung tissue models,
respectively) were prepared in a highly concentrated cell
suspension. Briefly, cells were liberated as described above,
collected, enumerated and pelleted by centrifugation. Supernatant
was removed and the cell pellet was resuspended in a small volume
of complete media, yielding a highly concentrated cell pellet of
1.times.10.sup.5 cells/.mu.L. This cell suspension was then stored
at 4.degree. C. until time of use.
[0254] Bioprinted tissue constructs were then fabricated into the
wells of a multi-well plate or onto the membrane of a cell culture
well insert (Transwell, BD). Multiple tissue types were created.
Multicellular NHLF or NHLF:EC bio-ink was used to bioprint a thick
interstitial tissue to recapitulate the wall of a small airway, and
subsequently layered with SAEC to provide the cognate epithelial
barrier layer. Vascular SMC or SMC:fibroblast bio-ink was used to
bioprint a thick interstitial tissue to recapitulate the blood
vessel wall, and subsequently layered with ECs to provide the
cognate endothelial barrier. hSC bio-ink was bioprinted in
conjunction with ECs into patches that either contained
interspersed endothelial networks or endothelial layers. Tissue
segments were fabricated with initial dimensions of 1.25 mm.times.8
mm.times.0.25 mm (W.times.L.times.H). Following bioprinting of the
lung construct or blood vessel wall segment, a concentrated cell
suspension was bioprinted on top of the previously-dispensed
bio-ink layer generating an additional defined layer of cells on
the surface of the first layer.
[0255] The bioprinted neotissues were then submerged in
serum-containing complete cell culture media and placed in a
standard humidified chamber, supplemented with 5% CO.sub.2 for
maturation. The bioprinted neotissues were then cultured and
stimulated with a relevant cytokine(s) for a predetermined period
of time, formalin-fixed, harvested, and processed for standard
histology and immunohistochemistry. The bioprinted tissues were
evaluated for characteristics such as, but not limited to for
tissue morphology, cell organization, extracellular matrix
production, cell proliferation, cell viability, and construct
integrity.
[0256] Cytokine stimulation was conducted by adding cytokine
directly to the culture media and incubating the bioprinted tissues
with the added protein to provide direct and prolonged cell access
to the proper stimulus. Dose-response experiments were conducted at
doses typically ranging from 0.1 to 100 ng/mL, dependent on the
ED50 of the experimental cytokine. For experiments in which
cytokine stimulation was conducted over more than 48 hours, media
was changed and fresh cytokine was added every 48 hours.
Results
[0257] Bioprinted neotissues containing physiologically-relevant
populations of cells were successfully stimulated with cytokines
that had been previously demonstrated to elicit cellular responses
in two-dimensional in vitro systems. The responses observed in the
bioprinted three-dimensional tissue constructs were observed to be
dose-dependent and unique to the cells within the bioprinted
construct (see, e.g., FIGS. 11, 15 and 16).
Example 19--Bioprinting of Co-Molded Functional Liver Tissue
Microstructure with Continuous Deposition
Preparation of 30% PF-127
[0258] A 30% PF-127 solution (w/w) was made using PBS. PF-127
powder was mixed with chilled PBS using a magnetic stir plate
maintained at 4.degree. C. Complete dissolution occurred in
approximately 48 hours.
Cell Preparation and Co-Printing of Mold and Fill
[0259] Three mL of PF-127 solution was aspirated into a 3 cc
reservoir using the bioprinter and with a 510 .mu.m dispense tip,
30% PF-127 solution was bioprinted onto a 6 well Transwell into a
single hexagon shape and layered sequentially 6 times.
[0260] A cell suspension, comprised of 7.8.times.10.sup.7 hepatic
cells (HepG2), was centrifuged at 1000 g for 6 minutes to create
the cell paste. Five .mu.L of cell paste was extruded through a 510
.mu.m needle to fill each of the triangular molds (see FIG. 17A).
The hexagon mold was incubated at room temperature for 15 minutes.
Three mL of media (DMEM supplemented with 10% FBS and 1.times.
penicillin, streptomycin and amphotericin B) was added to the well
with the Transwell supported above followed by incubation at
37.degree. C. and 5% CO.sub.2. Within 45 minutes the PF-127 mold
dissolved into the media leaving the molded hepatic bio-ink intact
to form a planar geometry of cells and void spaces (see FIG. 17B).
To remove residual PF-127 from the media, the Transwell was
transferred into a new well containing 3 mL of media and incubated
for two hours. This was repeated an additional 2 times for a total
media exchange of 9 mL over 6 hours.
[0261] Post 6 hours the Transwell was transferred to a new well
with no media and a cell suspension of 2.times.10.sup.6 cells, at a
ratio of human aortic endothelial cells at 90% and 10% hepatic
stellate cells, was dispensed to fill the voids created by the
dissolution of PF-127 mold. The hepatic constructs were incubated
for 15 minutes at room temperature. Following the 15 minute
incubation, 4 mL of media containing a ratio of 85% media (DMEM
supplemented with 10% FBS and 1.times. penicillin, streptomycin and
amphotericin B, to support the hepatic and stellate cells and 15%
M199 supplemented with 2% LSGS, 10% FBS, HEPES and 1.times.
penicillin, streptomycin and amphotericin B, to support the human
aortic endothelial cells). The construct was incubated at
37.degree. C. and 5% CO.sub.2 for 48 hours to form a contiguous
construct, with planar geometry comprising a lobular (triangular)
arrangement of hepatic parenchyma with intervening endothelial
cell-comprising tissue.
[0262] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein are suitably employed
in practicing the invention.
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