U.S. patent application number 14/933822 was filed with the patent office on 2016-05-05 for engineered three-dimensional skin tissues, arrays thereof, and methods of making the same.
The applicant listed for this patent is ORGANOVO, INC.. Invention is credited to Deborah Lynn Greene NGUYEN, Colin M. O'Neill, Sharon C. PRESNELL, Kelsey Nicole RETTING.
Application Number | 20160122723 14/933822 |
Document ID | / |
Family ID | 55851989 |
Filed Date | 2016-05-05 |
United States Patent
Application |
20160122723 |
Kind Code |
A1 |
RETTING; Kelsey Nicole ; et
al. |
May 5, 2016 |
ENGINEERED THREE-DIMENSIONAL SKIN TISSUES, ARRAYS THEREOF, AND
METHODS OF MAKING THE SAME
Abstract
Disclosed are bioprinted, three-dimensional, biological skin
tissues comprising: a dermal layer comprising dermal fibroblasts;
and an epidermal layer comprising keratinocytes, the epidermal
layer in contact with the dermal layer to form the
three-dimensional, engineered, biological skin tissue. Also
disclosed are arrays of engineered skin tissues and methods of
making engineered skin tissues.
Inventors: |
RETTING; Kelsey Nicole; (San
Diego, CA) ; O'Neill; Colin M.; (La Jolla, CA)
; NGUYEN; Deborah Lynn Greene; (San Diego, CA) ;
PRESNELL; Sharon C.; (Poway, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ORGANOVO, INC. |
San Diego |
CA |
US |
|
|
Family ID: |
55851989 |
Appl. No.: |
14/933822 |
Filed: |
November 5, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62140381 |
Mar 30, 2015 |
|
|
|
62075703 |
Nov 5, 2014 |
|
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Current U.S.
Class: |
435/366 ;
435/395 |
Current CPC
Class: |
A61L 27/60 20130101;
C12N 2503/06 20130101; C12N 2535/00 20130101; A61L 27/50 20130101;
C12N 2502/094 20130101; C12N 2533/54 20130101; C12N 2533/74
20130101; A61L 2300/64 20130101; C12N 5/0698 20130101; C12N
2502/091 20130101; C12N 2533/90 20130101; G01N 33/5044 20130101;
C12N 2502/1323 20130101; A61L 27/362 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071 |
Claims
1. A method of fabricating a three-dimensional, engineered,
biological skin tissue, the method comprising: a. preparing a
dermal bio-ink comprising dermal fibroblasts; b. preparing an
epidermal bio-ink comprising keratinocytes; c. depositing the
dermal bio-ink onto a surface; d. depositing the epidermal bio-ink
such that the epidermal bio-ink forms a layer on at least one
surface of the dermal bio-ink; and e. maturing the deposited
bio-ink in a cell culture media to allow the cells to cohere to
form the three-dimensional, engineered, biological skin tissue.
2. The method of claim 1, wherein the dermal bio-ink is deposited
by bioprinting, the bioprinting comprising extrusion of a
semi-solid or solid dermal bio-ink.
3. The method of claim 2, wherein the concentration of the dermal
bio-ink is between 5 and 500 million cells per mL.
4. The method of claim 1, wherein the dermal bio-ink is deposited
by bioprinting, the bioprinting comprising ink-jetting or spraying
a liquid dermal bio-ink.
5. The method of claim 4, wherein the concentration of the dermal
bio-ink is between 0.05 million and 50 million cells per mL.
6. The method of claim 1, wherein the epidermal bio-ink is
deposited by bioprinting, the bioprinting comprising extrusion of a
semi-solid or solid epidermal bio-ink.
7. The method of claim 6, wherein the concentration of the
epidermal bio-ink is between 5 and 500 million cells per mL.
8. The method of claim 1, wherein the epidermal bio-ink is
deposited by bioprinting, the bioprinting comprising ink jetting or
spraying a liquid epidermal bio-ink.
9. The method of claim 8, wherein the concentration of the
epidermal bio-ink is between 0.05 million and 50 million cells per
mL.
10. The method of claim 1, wherein the dermal bio-ink comprises
primary human fibroblasts.
11. The method of claim 10, wherein the dermal bio-ink consists
essentially of primary human fibroblasts.
12. The method of claim 1, wherein the epidermal bio-ink comprises
primary human keratinocytes.
13. The method of claim 1, wherein the epidermal bio-ink consists
essentially of primary human keratinocytes.
14. The method of claim 1, wherein the epidermal bio-ink comprises
melanocytes.
15. The method of claim 14, wherein the epidermal bio-ink consists
essentially of keratinocytes and melanocytes.
16. The method of claim 15, wherein the keratinocytes and
melanocytes are present in the epidermal bio-ink at a ratio of
about 90:10 to about 99:1 keratinocytes to melanocytes.
17. The method of claim 1, comprising depositing a plurality of
organoids into the deposited bio-ink, the organoids comprising:
sebocytes, glandular cells, or follicle cells.
18. The method of claim 1, comprising preparing a hypodermal
bio-ink, the hypodermal bio-ink comprising endothelial cells.
19. The method of claim 18, wherein the hypodermal bio-ink is
deposited on the surface prior to deposition of the dermal
bio-ink.
20. The method of claim 1, wherein either bio-ink comprises cancer
cells.
21. The method of claim 1, comprising depositing a test substance,
wherein a test substance is a substance under evaluation for its
ability to elicit a change in skin tissue compared to skin tissue
not treated with said substance.
22. The method of claim 21, wherein the test substance is deposited
on the apical surface of the epidermal layer.
23. The method of claim 1, wherein the epidermal bio-ink is
deposited on the at least one surface of the dermal bio-ink before
the dermal bio-ink is matured.
24. The method of claim 1, wherein the epidermal bio-ink is
deposited after the dermal bio-ink, and there is greater than 100
ms of delay between depositing the dermal and the epidermal
bio-ink.
25. The method of claim 1, wherein the deposited bio-inks are
matured for at least 24 hours.
26. The method of claim 1, wherein any of the deposited bio-inks
contain at least 70% live cells by volume at least 7 days post
deposition or any of the bio-inks, provided that the cells were not
treated with a test substance.
27. The method of claim 1, wherein the tissue comprises cells that
originated from two different donors.
28. The method of claim 1, wherein no mature tissue innervation,
perfusable lymphatic tissue, or perfusable vasculature were formed
during fabrication or maturation.
29. A method of fabricating a three-dimensional, engineered,
biological skin tissue, the method comprising: a. preparing a
dermal bio-ink comprising dermal fibroblasts, wherein the bio-ink
is a semi-solid or solid; wherein the concentration of the dermal
bio-ink is between 5 and 500 million cells per mL; b. preparing an
epidermal bio-ink comprising keratinocytes, wherein the
concentration of the epidermal bio-ink is between 0.05 million and
50 million cells per mL; c. depositing the dermal bio-ink onto a
surface by extrusion of the dermal bio-ink; d. depositing the
epidermal bio-ink by ink jetting or spraying the epidermal bio-ink
such that the epidermal bio-ink forms a layer on at least one
surface of the dermal bio-ink; and e. maturing the deposited
bio-ink in a cell culture media to allow the cells to cohere to
form the three-dimensional, engineered, biological skin tissue.
30. A method of fabricating a three-dimensional, engineered,
biological skin tissue, the method comprising: a. preparing a
dermal bio-ink comprising dermal fibroblasts, wherein the bio-ink
is a semi-solid or solid; wherein the concentration of the dermal
bio-ink is between 5 and 500 million cells per mL; b. preparing an
epidermal bio-ink comprising keratinocytes, wherein the bio-ink is
a semi-solid or solid; wherein the concentration of the epidermal
bio-ink is between 5 and 500 million cells per mL; c. depositing
the dermal bio-ink onto a surface by extrusion of the dermal
bio-ink; d. depositing the epidermal bio-ink by extrusion of the
epidermal bio-ink such that the epidermal bio-ink forms a layer on
at least one surface of the dermal bio-ink; and e. maturing the
deposited bio-ink in a cell culture media to allow the cells to
cohere to form the three-dimensional, engineered, biological skin
tissue.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Application Ser.
No. 62/075,703, filed Nov. 5, 2014 and U.S. Application Ser. No.
62/140,381, filed Mar. 30, 2015, the entire disclosures of which
are hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The pharmaceutical and cosmetic industries utilize skin
models for toxicology screening, barrier function analysis,
pigmentation studies, and models for corrosion, irritation,
inflammation, and infection.
SUMMARY OF THE INVENTION
[0003] Three-dimensional (3D) tissue models for human skin are
valuable as an alternative to animal models in both the
pharmaceutical and cosmetic industries, and clinically as a
therapeutic tissue engraftment. The full thickness human skin
models described herein contain a dermal compartment including
fibroblasts and connective tissue and an epidermal compartment
including stratified keratinocytes. Complexity of the model is
optionally increased by incorporating additional specialized cell
types, for example, melanocytes can be added into the epidermal
layer to model pigmentation. Further, complexity is added by the
addition of a hypodermal compartment comprising endothelial cells.
An advantage of utilizing skin equivalents developed in a
three-dimensional environment is that they are more physiologically
relevant than a two-dimensional environment. Cells in a
three-dimensional conformation may subsequently differentiate in a
different manner than cells cultured in a two-dimensional
monolayer, such as an alternative signaling pathway or through
different extracellular matrix interactions.
[0004] The engineered tissues described herein combine bio-ink
formulation technology with continuous deposition printing methods
and aerosol spray printing methods (such as ink jet) to create a
novel 3D tissue system to model skin. One major advantage of
bioprinting skin compared to existing skin models is the
reproducibility of an automated process. Another major advantage of
printing skin is the time frame in which a layered structure can be
generated. Current skin models often require a minimum of 3 weeks
to obtain a mature, layered structure. The bioprinting approaches
described herein overlay sheets of cells simultaneously to create
dermal and epidermal layers which are then allowed to mature and
differentiate for a defined period of time. The novel skin tissue
method presented in this disclosure exhibits a layered architecture
within a 12-day period.
[0005] In certain embodiments, described herein, is a
three-dimensional, engineered, biological skin tissue comprising: a
dermal layer comprising a dermal bio-ink, the dermal bio-ink
comprising fibroblasts; and an epidermal layer comprising epidermal
bio-ink, the epidermal bio-ink comprising keratinocytes, provided
the epidermal layer is in contact with the dermal layer to form the
three-dimensional, engineered, biological skin tissue. In certain
embodiments, the dermal layer consists essentially of dermal
fibroblasts. In certain embodiments, the epidermal layer consists
essentially of keratinocytes. In certain embodiments, the epidermal
layer consists essentially of primary keratinocytes. In certain
embodiments, the epidermal layer comprises primary keratinocytes.
In certain embodiments, the epidermal layer is substantially a
monolayer. In certain embodiments, the epidermal layer is
multilayered and comprises a plurality of layers of keratinocytes.
In certain embodiments, the epidermal layer was bioprinted onto the
dermal layer by aerosol spray deposition from a bioprinter. In
certain embodiments, the epidermal layer was bioprinted onto the
dermal layer immediately after bioprinting of the dermal layer. In
certain embodiments, the epidermal layer was bioprinted onto the
dermal layer after bioprinting and subsequent fusion of the dermal
layer. In certain embodiments, the epidermal layer is in continuous
contact with the dermal layer. In certain embodiments, greater than
90% of the epidermal layer is in contact with the dermal layer. In
certain embodiments, greater than 70% of the epidermal layer is in
contact with the dermal layer. In certain embodiments, greater than
50% of the epidermal layer is in contact with the dermal layer. In
certain embodiments, the epidermal layer is 20-500 .mu.m thick. In
certain embodiments, the epidermal layer is about 150 .mu.m thick.
In certain embodiments, the dermal layer is 10-1000 .mu.m thick. In
certain embodiments, the dermal layer is about 500 .mu.m thick. In
certain embodiments, the epidermal layer comprises an extrusion
compound. In certain embodiments, the dermal layer comprises an
extrusion compound. In certain embodiments, the epidermal layer
comprises melanocytes. In certain embodiments, the epidermal layer
consists essentially of keratinocytes and melanocytes. In certain
embodiments, the keratinocytes and melanocytes are present in the
epidermal layer at a ratio of about 99:1 to about 75:25
keratinocytes to melanocytes. In certain embodiments, the
keratinocytes and melanocytes are present in the epidermal layer at
a ratio of about 90:10 to about 99:1 keratinocytes to melanocytes.
In certain embodiments, the skin tissue comprises secretory cells.
In certain embodiments, the secretory cells comprise sebocytes. In
certain embodiments, the skin tissue comprises immune cells. In
certain embodiments, the immune cells comprise Langerhans cells. In
certain embodiments, the skin tissue comprises hair follicle stem
cells. In certain embodiments, the skin tissue comprises cancer
cells. In certain embodiments, the skin tissue comprises cells
derived from induced pluripotent stem cells or embryonic stem
cells. In certain embodiments, the skin tissue comprises a basal
layer in contact with the dermal layer and the epidermal layer,
wherein the basal layer comprises basal keratinocytes. In certain
embodiments, cells of the basal layer stain positive for KRT14
(CK14). In certain embodiments, the tissue is substantially free of
pre-formed scaffold. In certain embodiments, the fibroblasts and
keratinocytes are human cells. In certain embodiments, the dermal
layer comprises at least 30% live cells by volume. In certain
embodiments, the dermal layer comprises at least 70% live cells by
volume. In certain embodiments, the skin tissue comprises a
hypodermal layer ventral to the dermal layer, the hypodermal layer
comprising a hypodermal bio-ink, the hypodermal bio-ink comprising
endothelial cells. In certain embodiments, at least one bio-ink
comprises a plurality of organoids, the organoids comprising
glandular cells or follicle cells. In certain embodiments, the
epidermal bio-ink comprises 0.05 to 50 million cells per ml. In
certain embodiments, the epidermal bio-ink comprises 5 to 500
million cells per ml. In certain embodiments, the epidermal bio-ink
comprises about 150 million cells per ml. In certain embodiments,
the dermal bio-ink comprises 0.05 to 50 million cells per ml. In
certain embodiments, the dermal bio-ink comprises 5 to 500 million
cells per ml. In certain embodiments, the dermal bio-ink comprises
about 150 million cells per ml. In certain embodiments, the skin
tissue comprises a test substance, wherein a test substance is a
substance under evaluation for its ability to elicit a change in
said skin tissue compared to skin tissue not treated with said
substance. In certain embodiments, the test substance is
homogenously present throughout the dermal layer, the epidermal
layer or both the dermal and epidermal layer. In certain
embodiments, the test substance is heterogeneously present
throughout the dermal layer, the epidermal layer or both the dermal
and epidermal layer. In certain embodiments, the test substance is
in contact with the apical side of the epidermal layer. In certain
embodiments, the test substance is between the epidermal and dermal
layers. In certain embodiments, the test substance is between the
dermal layer and the printing surface. In certain embodiments, the
test substance is in contact with the lateral surface. In certain
embodiments, the test substance is within a discreet compartment
embedded within the dermal layer, the epidermal layer or both the
dermal and epidermal layer. In certain embodiments, the skin tissue
comprises a therapeutic substance. In certain embodiments, the
therapeutic substance is homogenously present throughout the dermal
layer, the epidermal layer or both the dermal and epidermal layer.
In certain embodiments, the therapeutic substance is
heterogeneously present throughout the dermal layer, the epidermal
layer or both the dermal and epidermal layer. In certain
embodiments, the therapeutic substance is in contact with the
apical side of the epidermal layer. In certain embodiments, the
therapeutic substance is between the epidermal and dermal layers.
In certain embodiments, the therapeutic substance is between the
dermal layer and the printing surface. In certain embodiments, the
therapeutic substance is in contact with the lateral surface. In
certain embodiments, the therapeutic substance is within a discreet
compartment embedded within the dermal layer, the epidermal layer
or both the dermal and epidermal layer. In certain embodiments, the
deposition of at least one layer was temporally delayed after the
deposition of the previous layer. In certain embodiments, the
temporal delay is greater than 10 milliseconds. In certain
embodiments, the skin tissue is a genetic chimera. In certain
embodiments, the skin tissue is a species chimera. In certain
embodiments, the skin tissue is configured in an array to
facilitate an in vitro assay, drug-screening assay, or a cosmetic
assay. In certain embodiments, the array is configured to allow at
least 20 .mu.m of space between each tissue.
[0006] In certain embodiments, described herein, is a method of
fabricating a three-dimensional, engineered, biological skin
tissue, the method comprising: preparing a dermal bio-ink
comprising dermal fibroblasts; preparing an epidermal bio-ink
comprising keratinocytes; depositing the dermal bio-ink on a
surface; depositing the epidermal bio-ink such that the epidermal
bio-ink forms a layer on at least one surface of the dermal
bio-ink; and maturing the deposited bio-ink in a cell culture media
to allow the cells to cohere to form the three-dimensional,
engineered, biological skin tissue. In certain embodiments, the
dermal bio-ink is deposited by extrusion bioprinting. In certain
embodiments, the epidermal bio-ink is deposited by aerosol spray
bioprinting. In certain embodiments, the dermal bio-ink is
deposited by aerosol spray bioprinting. In certain embodiments, a
support material is deposited by aerosol spray bioprinting. In
certain embodiments, the dermal bio-ink is at least 30% live cells
by volume. In certain embodiments, the epidermal bio-ink is at
least 30% live cells by volume. In certain embodiments, the
epidermal bio-ink comprises primary keratinocytes. In certain
embodiments, the epidermal bio-ink consists essentially of primary
keratinocytes. In certain embodiments, the epidermal bio-ink
comprises melanocytes. In certain embodiments, the epidermal
bio-ink consists essentially of keratinocytes and melanocytes. In
certain embodiments, the dermal bio-ink or the epidermal bio-ink
comprises cancer cells. In certain embodiments, the dermal bio-ink
or the epidermal bio-ink comprises cells derived from induced
pluripotent stem cells or embryonic stem cells. In certain
embodiments, the method comprises depositing a plurality of
organoids into the deposited bio-ink, the organoids comprising
glandular cells or follicle cells. In certain embodiments, the
method comprises preparing a hypodermal bio-ink, the hypodermal
bio-ink comprising endothelial cells. In certain embodiments, the
hypodermal bio-ink is deposited on the surface on at least one
surface followed by deposition of the dermal bio-ink. In certain
embodiments, the epidermal bio-ink comprises 0.05 to 50 million
cells per ml In certain embodiments, the epidermal bio-ink
comprises 5 to 500 million cells per ml. In certain embodiments,
the epidermal bio-ink comprises about 150 million cells per ml. In
certain embodiments, the dermal bio-ink comprises 0.05 to 50
million cells per ml. In certain embodiments, the dermal bio-ink
comprises 5 to 500 million cells per ml. In certain embodiments,
the dermal bio-ink comprises about 150 million cells per ml. In
certain embodiments, the dermal bio-ink comprises an extrusion
compound. In certain embodiments, the epidermal bio-ink comprises
an extrusion compound. In certain embodiments, the keratinocytes
and melanocytes are present in the epidermal bio-ink at a ratio of
about 99:1 to about 75:25 keratinocytes to melanocytes. In certain
embodiments, the keratinocytes and melanocytes are present in the
epidermal bio-ink at a ratio of about 90:10 to about 99:1
keratinocytes to melanocytes. In certain embodiments, either
bio-ink comprises secretory cells. In certain embodiments, the
secretory cells comprise sebocytes. In certain embodiments, either
bio-ink comprises immune cells. In certain embodiments, the immune
cells comprise Langerhans cells. In certain embodiments, either
bio-ink comprises hair follicle stem cells. In certain embodiments,
either bio-ink comprises cancer cells. In certain embodiments,
either bio-ink comprises cells derived from induced pluripotent
stem cells or embryonic stem cells. In certain embodiments, the
method comprises depositing a basal layer in contact with the
dermal layer and the epidermal layer, wherein the basal layer
comprises a bio-ink comprising basal keratinocytes. In certain
embodiments, the tissue is not deposited on a scaffold. In certain
embodiments, the fibroblasts and keratinocytes are human cells. In
certain embodiments, the method comprises depositing a test
substance, wherein a test substance is a substance under evaluation
for its ability to elicit a change in skin tissue compared to skin
tissue not treated with said substance. In certain embodiments, the
test substance is homogenously deposited throughout the dermal
layer, the epidermal layer or both the dermal and epidermal layer.
In certain embodiments, the test substance is heterogeneously
deposited throughout the dermal layer, the epidermal layer or both
the dermal and epidermal layer. In certain embodiments, the test
substance is deposited in contact with the apical side of the
epidermal layer. In certain embodiments, the test substance is
deposited between the epidermal and dermal layers. In certain
embodiments, the test substance is deposited between the dermal
layer and the printing surface. In certain embodiments, the test
substance is deposited in contact with the lateral surface. In
certain embodiments, the test substance is deposited within a
discreet compartment embedded within the dermal layer, the
epidermal layer or both the dermal and epidermal layer. In certain
embodiments, the method comprises depositing a therapeutic
substance. In certain embodiments, the therapeutic substance is
homogenously deposited throughout the dermal layer, the epidermal
layer or both the dermal and epidermal layer. In certain
embodiments, the therapeutic substance is heterogeneously deposited
throughout the dermal layer, the epidermal layer or both the dermal
and epidermal layer. In certain embodiments, the therapeutic
substance is deposited in contact with the apical side of the
epidermal layer. In certain embodiments, the therapeutic substance
is deposited between the epidermal and dermal layers. In certain
embodiments, the therapeutic substance is deposited between the
dermal layer and the printing surface. In certain embodiments, the
therapeutic substance is deposited in contact with the lateral
surface. In certain embodiments, the therapeutic substance is
deposited within a discreet compartment embedded within the dermal
layer, the epidermal layer or both the dermal and epidermal layer.
In certain embodiments, the method comprises a temporal delay in
the deposition of the epidermal bio-ink onto the dermal bio-ink. In
certain embodiments, the delay is greater than 10 milliseconds.
[0007] In another aspect described herein, is a three-dimensional,
engineered, biological multi-tissue system comprising: a first
engineered tissue, the first engineered tissue an engineered skin
tissue comprising: a dermal layer comprising a dermal bio-ink, the
dermal bio-ink comprising dermal fibroblasts; and an epidermal
layer comprising an epidermal bio-ink, the epidermal bio-ink
comprising keratinocytes, the epidermal layer in contact with the
dermal layer to form the engineered skin tissue; and a second
engineered tissue, the second engineered tissue not a skin tissue,
the second engineered tissue in physical or fluidic contact with
the engineered skin tissue to form the multi-tissue system, wherein
at least one component of the second tissue was bioprinted. In
certain embodiments, at least one component of the first engineered
tissue was bioprinted. In certain embodiments, at least one
component of the second engineered tissue was bioprinted. In
certain embodiments, the second engineered tissue is a liver
tissue, a kidney tissue, a bone tissue, a lung tissue, a vascular
tissue, a brain tissue, an intestinal tissue, a stomach tissue, or
an esophageal tissue. In certain embodiments, the engineered skin
tissue is substantially free of pre-formed scaffold at the time of
use. In certain embodiments, the second engineered tissue is
substantially free of pre-formed scaffold at the time of use.
[0008] In certain embodiments, described herein, is a
three-dimensional, engineered, biological skin tissue comprising a
dermis, the dermis comprising dermal fibroblasts; provided that the
dermis was bioprinted from a dermal bio-ink and fused to form the
three-dimensional, engineered, biological skin tissue.
[0009] In another aspect described herein, is a three-dimensional,
engineered, biological skin tissue comprising an epidermis, the
epidermis comprising keratinocytes; provided that the epidermis was
bioprinted from an epidermal bio-ink and fused to form the
three-dimensional, engineered, biological skin tissue.
[0010] In another aspect described herein, is a method of
fabricating a three-dimensional, engineered, biological skin
tissue, the method comprising: preparing a dermal bio-ink
comprising dermal fibroblasts; preparing an epidermal bio-ink
comprising keratinocytes; depositing the dermal bio-ink on a
surface; depositing the epidermal bio-ink such that the epidermal
bio-ink forms a layer on at least one surface of the dermal
bio-ink, wherein the deposition of the epidermal bio-ink occurs
greater than 10 milliseconds and less than 21 days after the after
deposition of the dermal bio-ink.
[0011] In another aspect, disclosed herein are three-dimensional,
engineered, biological skin tissues comprising a dermis, the dermis
comprising dermal fibroblasts; provided that the dermis was
bioprinted from a dermal bio-ink and fused to form the
three-dimensional, engineered, biological skin tissue.
[0012] In another aspect, disclosed herein are three-dimensional,
engineered, biological skin tissues comprising an epidermis, the
epidermis comprising keratinocytes; provided that the epidermis was
bioprinted from an epidermal bio-ink and fused to form the
three-dimensional, engineered, biological skin tissue.
[0013] In another aspect, disclosed herein are three-dimensional,
engineered, biological skin tissues comprising a test substance,
wherein a test substance is a substance under evaluation for its
ability to elicit a change in said skin tissue compared to skin
tissue not treated with said substance. The test substance can be
homogenously or heterogeneously present throughout the dermal
layer, the epidermal layer or both the dermal and epidermal layer.
The test substance can be in contact with the apical side of the
epidermal layer, between the epidermal and dermal layers, between
the dermal layer and the printing surface, within a discreet
compartment embedded within the dermal layer, the epidermal layer
or both the dermal and epidermal layer, or in contact with any
lateral surface of the tissue.
[0014] In another aspect, disclosed herein are three-dimensional,
engineered, biological skin tissues comprising a therapeutic
substance. The therapeutic substance can be homogenously or
heterogeneously present throughout the dermal layer, the epidermal
layer or both the dermal and epidermal layer. The therapeutic
substance can be in contact with the apical side of the epidermal
layer, between the epidermal and dermal layers, between the dermal
layer and the printing surface, within a discreet compartment
embedded within the dermal layer, the epidermal layer or both the
dermal and epidermal layer, or in contact with any lateral surface
of the tissue.
[0015] In another aspect, disclosed herein are three-dimensional,
engineered, biological skin tissues constructed with a temporal
delay in deposition between the dermal and epidermal layers.
[0016] In another aspect, disclosed herein are methods of
fabricating a three-dimensional, engineered, biological skin
tissue, the method comprising: preparing a dermal bio-ink
comprising dermal fibroblasts; preparing an epidermal bio-ink
comprising keratinocytes; depositing the dermal bio-ink onto a
surface; depositing the epidermal bio-ink such that the epidermal
bio-ink forms a layer on at least one surface of the dermal
bio-ink; and maturing the deposited bio-ink in a cell culture media
to allow the cells to cohere to form the three-dimensional,
engineered, biological skin tissue. In certain embodiments, the
dermal bio-ink is deposited by bioprinting, the bioprinting
comprising extrusion of a semi-solid or solid dermal bio-ink. In
certain embodiments, the concentration of the dermal bio-ink is
between 5 and 500 million cells per mL. In certain embodiments, the
dermal bio-ink is deposited by bioprinting, the bioprinting
comprising ink jetting or spraying a liquid dermal bio-ink. In
certain embodiments, the concentration of the dermal bio-ink is
between 0.05 million and 50 million cells per mL. In certain
embodiments, the epidermal bio-ink is deposited by bioprinting, the
bioprinting comprising extrusion of a semi-solid or solid epidermal
bio-ink. In certain embodiments, the concentration of the epidermal
bio-ink is between 5 and 500 million cells per mL. In certain
embodiments, the epidermal bio-ink is deposited by bioprinting, the
bioprinting comprising ink jetting or spraying a liquid epidermal
bio-ink. In certain embodiments, the concentration of the epidermal
bio-ink is between 0.05 million and 50 million cells per mL. In
certain embodiments, the dermal bio-ink comprises primary human
fibroblasts. In certain embodiments, the dermal bio-ink consists
essentially of primary human fibroblasts. In certain embodiments,
the epidermal bio-ink comprises primary human keratinocytes. In
certain embodiments, the epidermal bio-ink consists essentially of
primary human keratinocytes. In certain embodiments, the epidermal
bio-ink comprises melanocytes. In certain embodiments, the
epidermal bio-ink consists essentially of keratinocytes and
melanocytes. In certain embodiments, the keratinocytes and
melanocytes are present in the epidermal bio-ink at a ratio of
about 90:10 to about 99:1 keratinocytes to melanocytes. In certain
embodiments, the method comprises depositing a plurality of
organoids into the deposited bio-ink, the organoids comprising:
sebocytes, glandular cells, or follicle cells. In certain
embodiments, the method comprises preparing a hypodermal bio-ink,
the hypodermal bio-ink comprising endothelial cells. In certain
embodiments, the hypodermal bio-ink is deposited on the surface
prior to deposition of the dermal bio-ink. In certain embodiments,
either bio-ink comprises cancer cells. In certain embodiments, the
method comprises depositing a test substance, wherein a test
substance is a substance under evaluation for its ability to elicit
a change in skin tissue compared to skin tissue not treated with
said substance. In certain embodiments, the test substance is
deposited on the apical surface of the epidermal layer. In certain
embodiments, the epidermal bio-ink is deposited on the at least one
surface of the dermal bio-ink before the dermal bio-ink is matured.
In certain embodiments, the epidermal bio-ink is deposited after
the dermal bio-ink, and there is greater than 100 ms of delay
between depositing the dermal and the epidermal bio-ink. In certain
embodiments, the deposited bio-inks are matured for at least 24
hours. In certain embodiments, any of the deposited bio-inks
contain at least 70% live cells by volume at least 7 days post
deposition or any of the bio-inks, provided that the cells were not
treated with a test substance. In certain embodiments, the tissue
comprises cells that originated from two different donors. In
certain embodiments, mature tissue innervation, perfusable
lymphatic tissue, and/or perfusable vasculature were not formed
during fabrication or maturation and are absent from the engineered
tissue.
[0017] In another aspect, disclosed herein are methods of
fabricating a three-dimensional, engineered, biological skin
tissue, the method comprising: preparing a dermal bio-ink
comprising dermal fibroblasts, wherein the bio-ink is a semi-solid
or solid; wherein the concentration of the dermal bio-ink is
between 5 and 500 million cells per mL; preparing an epidermal
bio-ink comprising keratinocytes, wherein the concentration of the
epidermal bio-ink is between 0.05 million and 50 million cells per
mL; depositing the dermal bio-ink onto a surface by extrusion of
the dermal bio-ink; depositing the epidermal bio-ink by ink jetting
or spraying the epidermal bio-ink such that the epidermal bio-ink
forms a layer on at least one surface of the dermal bio-ink; and
maturing the deposited bio-ink in a cell culture media to allow the
cells to cohere to form the three-dimensional, engineered,
biological skin tissue.
[0018] In another aspect, disclosed herein are methods of
fabricating a three-dimensional, engineered, biological skin
tissue, the method comprising: preparing a dermal bio-ink
comprising dermal fibroblasts, wherein the bio-ink is a semi-solid
or solid; wherein the concentration of the dermal bio-ink is
between 5 and 500 million cells per mL; preparing an epidermal
bio-ink comprising keratinocytes, wherein the bio-ink is a
semi-solid or solid; wherein the concentration of the epidermal
bio-ink is between 5 and 500 million cells per mL; depositing the
dermal bio-ink onto a surface by extrusion of the dermal bio-ink;
depositing the epidermal bio-ink by extrusion of the epidermal
bio-ink such that the epidermal bio-ink forms a layer on at least
one surface of the dermal bio-ink; and maturing the deposited
bio-ink in a cell culture media to allow the cells to cohere to
form the three-dimensional, engineered, biological skin tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0020] FIG. 1 shows a non-limiting example of a schematic structure
diagram depicting optional features of the engineered skin tissues
described herein.
[0021] FIG. 2 shows a non-limiting example of a diagram
illustrating the stratified epidermal layers seen in normal skin
tissues and exemplary biomarkers suitable to visualize specific
layers.
[0022] FIG. 3 shows a non-limiting example of a schematic flow
diagram depicting a method of fabricating a skin tissue; in this
case, a method of fabricating a layered engineered skin tissue
including depositing, using continuous deposition techniques, a
layer of dermal cells onto a surface and depositing, using
continuous deposition bioprinting techniques, a layer of epidermal
cells onto the layer of dermal cells.
[0023] FIG. 4 shows a non-limiting example of a macroscale
photograph of an engineered skin tissue; in this case, an
engineered skin tissue including dermal and epidermal layers,
deposited by continuous deposition bioprinting techniques, at 48
hours post printing.
[0024] FIG. 5 shows a non-limiting example of a schematic flow
diagram depicting a method of fabricating a skin tissue; in this
case, a method of fabricating a layered engineered skin tissue
including depositing, using continuous deposition bioprinting
techniques, a layer of dermal cells onto a surface and depositing,
using aerosol spray bioprinting techniques, using a monolayer (A),
or a plurality of layers of epidermal cells (B) onto the layer of
dermal cells. In (C) another embodiment is shown, a layer of dermal
cells is bioprinted onto a surface using continuous deposition
bioprinting techniques followed by addition of a layer of
extracellular matrix, followed by addition of a monolayer or a
plurality of layers of epidermal cells.
[0025] FIG. 6 shows a non-limiting example of a schematic flow
diagram depicting a method of fabricating a skin tissue; in this
case, a method of fabricating a layered engineered skin tissue
including depositing, using ink jet deposition bioprinting
techniques, to embed a layer of dermal cells into a surface and
depositing, using aerosol spray bioprinting or continuous
deposition techniques, epidermal cells onto the layer of dermal
cells.
[0026] FIG. 7 shows a non-limiting example of an experimental
design; in this case, an experimental design depicting a variety of
bioprinting techniques used to achieve the engineered skin tissues
described herein.
[0027] FIG. 8 shows non-limiting examples of macroscale photographs
of an engineered skin tissue; in this case, an engineered skin
tissue including a dermal layer, immediately post printing (A) and
1 day post printing (B).
[0028] FIG. 9 shows non-limiting examples of macroscale photographs
of an engineered skin tissue; in this case, an engineered
full-thickness skin tissue, immediately post printing of the dermal
layer (A), 24 hours post printing of the dermal layer (B),
immediately post printing of the epidermal layer (C), 24 hours post
printing of the epidermal layer (D), 96 hours post exposure of the
tissue to the air-liquid interface (E), and 216 hours post exposure
of the tissue to the air-liquid interface (F).
[0029] FIG. 10 shows non-limiting examples of photomicrographs of
engineered skin tissues; in this case, photomicrographs depicting
H&E staining (A and B) and immunohistochemistry for
visualization of CK14 (C and D) of the tissues of Example 1 at day
2 post printing.
[0030] FIG. 11 shows non-limiting examples of photomicrographs of
engineered skin tissues; in this case, photomicrographs depicting
H&E staining (A, C, E, G, I, and K) and immunohistochemistry
for visualization of CK14 (B, D, F, H, J, and L) of the tissues of
Example 1 at day 2 post printing (A-D), day 4 post printing (E-H),
and day 8 post printing (I-L).
[0031] FIG. 12 shows non-limiting examples of photomicrographs of
engineered skin tissues; in this case, photomicrographs depicting
H&E staining (A) and immunohistochemistry for visualization of
CK14 (B) of epithelial layers deposited by aerosol spray
bioprinting directly onto a surface.
[0032] FIG. 13 shows non-limiting examples of photomicrographs of
engineered skin tissues; in this case, photomicrographs depicting
H&E staining of two of the tissues of Example 2 at day 12 post
printing (first tissue: A; second tissue B) (arrows indicate
distinct basal layer).
[0033] FIG. 14 shows non-limiting examples of photomicrographs of
engineered skin tissues; in this case, photomicrographs depicting
H&E staining (A, C, E, and G) and immunohistochemistry for
visualization of CK14 (B, D, F, and H) of two of the tissues of
Example 2 at day 12 post printing (first tissue: A-D; second tissue
E-H).
[0034] FIG. 15 shows non-limiting examples of photomicrographs of
engineered skin tissues; in this case, photomicrographs depicting
immunohistochemistry for visualization of CK5/IVL/Dapi (A and C)
and CK10/Dapi (B and D) of a third tissue of Example 2 at day 12
post printing.
[0035] FIG. 16 shows non-limiting examples of photomicrographs of
engineered skin tissues; in this case, photomicrographs depicting a
comparison of tissues bioprinted using different methodologies
(first tissue at day 10 (A); second tissue at day 12 (B)).
[0036] FIG. 17 shows exemplary experimental data on gene expression
within the engineered skin tissues described herein; in this case,
a gene expression data for collagen (COL1 and COL4), filaggrin
(FLG), and cytokeratin (CK1 and CK10).
[0037] FIG. 18 shows the effect that dermal tissue has on epidermal
organization and differentiation. (A) Is a schematic of the
experiment. (B) shows a macroscopic view of the printed tissue. (C
and F) low magnification of H&E stained cells printed without
(C) or with (F) dermal paste (bio-ink). (D and G) higher
magnification of H&E stained cells printed without (D) or with
(G) dermal paste (bio-ink). Distinct layers of differentiated
keratinocytes are visualized by simultaneously staining for a basal
cell marker CK5 (green) and involucrin (IVL, red), a later stage
differentiation marker of granular and cornified keratinocytes in
cells printed without (E) or with dermal paste (bio-ink) (H).
[0038] FIG. 19 histological analysis of bioprinted skin tissue at
day 12. Shown is H&E staining (A), staining for CK5/IVL (B),
CK10 (C), Trichrome stain (D), PCNA and Collagen (E) and TUNEL
staining (F).
[0039] FIG. 20 shows gene expression analysis of bioprinted skin
tissue at day 12.
[0040] FIG. 21 Shows LDH activity (A), IL-1.alpha. production (B)
and alamar blue assay (C) of bioprinted skin tissues treated with
1% Triton X100.TM..
[0041] FIG. 22 shows histological analysis of bioprinted skin
tissues treated with 1% Triton X-100.TM. (G, H, I, J, K, L)
compared to PBS treated controls (A, B C, D, E, F). Cells were
stained for H&E (A and G), CK5 (green) and IVL (red) (B and H),
CK10 (C and I), Trichrome (D and J), PCNA (green) and Collagen
(red) (E and K) and TUNEL (F and L).
[0042] FIG. 23 shows gene expression analysis of dermal markers
from bioprinted skin tissue after treatment with 1% Triton X100.TM.
and PBS for 48 hours.
[0043] FIG. 24 shows ILIA gene expression from bioprinted skin
tissue after 48 hours after treatment with 1% Triton X-100.TM. or
PBS.
[0044] FIG. 25 shows LDH activity (A), IL-1.alpha. production (B)
and alamar blue assay (C) of bioprinted skin tissues treated with
1% Triton X-100.TM. or 5% SDS.
[0045] FIG. 26 shows non-limiting examples of printing
configurations to apply a test substance to printed tissue.
[0046] FIG. 27 shows non-limiting examples of printing
configurations to apply a therapeutic substance to printed
tissue.
[0047] FIG. 28 shows two non-limiting examples of
three-dimensional, engineered, biological skin tissues printed in
Example 6. Tissue example 1 (A, C, E, G) and Tissue example 2 (B,
D, F, H). The tissue were stained for H&E (A and B); B (CK5),
(green), and Involucrin (IVL) (red) (C and D); Collagen 4 (COL4) (E
and F); and Collagen 7 (COL7) (G and H).
[0048] FIG. 29 shows H&E staining of a natural tissue (A), and
a non-limiting example of three-dimensional, engineered, biological
skin tissue (B).
DETAILED DESCRIPTION OF THE INVENTION
[0049] Described herein, in certain embodiments, are
three-dimensional, engineered, biological skin tissues comprising:
a dermal layer comprising dermal fibroblasts; and an epidermal
layer comprising keratinocytes, the epidermal layer in contact with
the dermal layer to form the three-dimensional, engineered,
biological skin tissue; provided that the dermal layer was
bioprinted from a dermal bio-ink, the epidermal layer was
bioprinted from an epidermal bio-ink, or both the dermal layer and
the epidermal layer were bioprinted from their respective
bio-inks.
[0050] Also described herein, in certain embodiments, are arrays of
three-dimensional, engineered, biological skin tissues, each skin
tissue comprising: a dermal layer comprising dermal fibroblasts;
and an epidermal layer comprising keratinocytes, the epidermal
layer in contact with the dermal layer to form the
three-dimensional, engineered, biological skin tissue; provided
that the dermal layer, the epidermal layer, or both the dermal
layer and the epidermal layer were bioprinted; provided that the
array is adapted for use in screening assays.
[0051] Also described herein, in certain embodiments, are methods
of fabricating a three-dimensional, engineered, biological skin
tissue, the method comprising: preparing a dermal bio-ink
comprising dermal fibroblasts; preparing an epidermal bio-ink
comprising keratinocytes; depositing the dermal bio-ink on a
surface; depositing the epidermal bio-ink such that the epidermal
bio-ink forms a layer on at least one surface of the dermal
bio-ink; and maturing the deposited bio-ink in a cell culture media
to allow the cells to cohere to form the three-dimensional,
engineered, biological skin tissue.
[0052] Also described herein, in certain embodiments, are
three-dimensional, engineered, biological multi-tissue systems
comprising: a first engineered tissue, the first engineered tissue
an engineered skin tissue comprising: a dermal layer comprising
dermal fibroblasts; and an epidermal layer comprising
keratinocytes, the epidermal layer in contact with the dermal layer
to form the engineered skin tissue; wherein at least one component
of the skin tissue was bioprinted; and a second engineered tissue,
the second engineered tissue not a skin tissue, the second
engineered tissue in physical or fluidic contact with the
engineered skin tissue to form the multi-tissue system, wherein at
least one component of the second tissue was bioprinted.
[0053] Also described herein, in certain embodiments, are
three-dimensional, engineered, biological skin tissues comprising a
dermis, the dermis comprising dermal fibroblasts; provided that the
dermis was bioprinted from a dermal bio-ink and fused to form the
three-dimensional, engineered, biological skin tissue.
[0054] Also described herein, in certain embodiments, are
three-dimensional, engineered, biological skin tissues comprising
an epidermis, the epidermis comprising keratinocytes; provided that
the epidermis was bioprinted from an epidermal bio-ink and fused to
form the three-dimensional, engineered, biological skin tissue.
[0055] Also described herein, in certain embodiments, are methods
of fabricating a three-dimensional, engineered, biological skin
tissues, the methods comprising: preparing a dermal bio-ink
comprising dermal fibroblasts; preparing an epidermal bio-ink
comprising keratinocytes; depositing the dermal bio-ink on a
surface; depositing the epidermal bio-ink such that the epidermal
bio-ink forms a layer on at least one surface of the dermal
bio-ink, wherein the deposition of the epidermal bio-ink occurs
greater than 10 milliseconds after deposition of the dermal
bio-ink.
CERTAIN DEFINITIONS
[0056] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. 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. Any reference to "or" herein is
intended to encompass "and/or" unless otherwise stated.
[0057] As used herein, "tissue" means an aggregate of cells.
[0058] 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.
[0059] 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.).
[0060] As used herein, "compartment" means an association of cells
or extracellular matrix components cohered to create a distinct
type or sub-type of a tissue such as epidermal, dermal, hypodermal
or basal; or a specialized organoid such as a follicle. An organoid
is an association of cells that perform a dedicated function (a
hair follicle for example). A compartment can comprise a layered
geometry, but also cell conglomerates with any geometric or
irregular shape.
[0061] As used herein, "layer" means an association of cells or
extracellular matrix components in X and Y planes that is multiple
cells thick. In some embodiments, the engineered skin tissues
describe herein include one layer. In other embodiments, the
engineered skin tissues describe herein include a plurality of
layers. In various embodiments, a layer forms a contiguous,
substantially contiguous, or non-contiguous sheet of cells and/or
extracellular matrix components. In some embodiments, each layer of
an engineered skin tissue described herein comprises multiple cells
in the X, Y, and Z axes. In some embodiments, an engineered skin
tissue described herein comprises only of layers of cells. In other
embodiments, a layer of extracellular matrix components can be
printed between layers of cells.
[0062] As used herein, "bio-ink" means a liquid, semi-solid, or
solid composition for use in bioprinting. In some embodiments,
bio-ink comprises cell solutions, cell aggregates, cell-comprising
gels, multicellular bodies, or tissues. In some embodiments, the
bio-ink can be a solid or semi-solid. In some embodiments, the
bio-ink additionally comprises non-cellular materials that provide
specific biomechanical properties that enable bioprinting. In some
embodiments, the bio-ink comprises an extrusion compound. In some
cases, the extrusion compound is engineered to be removed after the
bioprinting process. In other embodiments, at least some portion of
the extrusion compound remains entrained with the cells
post-printing and is not removed.
[0063] As used herein, "bioprinting" means utilizing
three-dimensional, precise deposition of cells (e.g., cell
solutions, cell-containing gels, cell suspensions, cell pastes,
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). Bioprinting encompasses methods
compatible with printing living cells such as extrusion in
continuous and/or discontinuous fashion. Extrusion in this context
means forcing a semi-solid or solid bio-ink through an orifice,
wherein the bio-ink retains its shape to a degree and for a time
period after being forced through the orifice. Bioprinting also
encompasses aerosol spray methods where cells are applied by
ejecting a substantially low viscosity liquid in a mist, spray, or
droplets onto a surface. Suitable bioprinters include Novogen
Bioprinters.RTM. from Organovo, Inc. (San Diego, Calif.).
[0064] 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 not able to be removed from the tissue
without damage/destruction of said tissue. 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 pre-formed 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."
[0065] 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."
[0066] As used herein, "test substance" refers to any biological,
chemical or physical substance under evaluation for its ability to
elicit a change in said skin tissue compared to skin tissue not
treated with said substance. A non-limiting example of a change in
skin tissue could be an allergic reaction, a toxic reaction, an
irritation reaction; a change that is measured by a defined
molecular state such as a change in mRNA levels or activity,
changes in protein levels, changes in protein modification or
epigenetic changes; or a change that results in a measurable
cellular outcome such as a change in proliferation, apoptosis, cell
viability, cell division, cell motility, cytoskeletal
rearrangements, chromosomal number or composition. Test substances
include, but are not limited to; chemical compositions containing
an active or inactive ingredient, either in whole, in part,
isolated, or purified; physical stressors such as light, UV light,
mechanical stress, heat, or cold; biological agents such as
bacteria, viruses, parasites, or fungi. "Test substance" also
refers to a plurality of substances mixed or applied
separately.
[0067] As used herein, "therapeutic substance" refers to any
composition containing an active ingredient, which can be used to
treat a condition in a subject. Examples of active ingredients
include but are not limited to antibiotics, antivirals,
antifungals, anti-inflammatories, immunosuppressants, analgesics,
opiates, vasoconstrictors, vasodilators, steroids, vitamin mixtures
or supplement mixtures. "Therapeutic substance" also refers to a
plurality of substances mixed or applied separately. A therapeutic
substance can also be any substance used to protect skin or promote
its attachment and ability to thrive at a site of engraftment.
These include but are not limited to skin protectants,
moisturizers, adhesives (biodegradable or non-biodegradable),
physical barriers (biodegradable or non-biodegradable), porous
membranes, or non-porous membranes.
[0068] As used herein, "toxicology" refers to the assessment of
any, biological, chemical or physical agent for harm when contacted
with a tissue. The dosage amounts of agents for toxicology testing
can include dosage ranges that are greater than or less than what
would be considered a recommended, physiological or therapeutic
dose.
[0069] As used herein, "use" encompasses a variety of possible uses
of the tissue which will be appreciated by one skilled in the art.
These uses include by way of non-limiting example; implantation or
engraftment of the engineered tissue into or onto a subject;
inclusion of the tissue in a biological assay for the purposes of
biological, biotechnological or pharmacological discovery;
toxicology testing, including teratogen testing; pharmacology
testing, including testing to determine pharmacokinetics and drug
metabolism and absorption and skin penetration, cosmetic testing,
including testing to determine sensitization, potential to cause
irritation or corrosion of any layer of the dermis, to any test
chemical or non-chemical agent including ultraviolet light. "Use"
can also refer to the process of maturation, or tissue cohesion, in
vitro after bioprinting.
[0070] As used herein, "substantially free" means less than 2.0%,
less than 1.0%, less than 0.1%, only trace amounts, or entirely
free of the indicated substance, tissue-type, cell-type, or
structure.
Engineered, Three-Dimensional Skin Tissues
[0071] Three-dimensional skin tissues disclosed herein represent an
improvement to the state of the art. Tissue models for human skin
are valuable in both the cosmetic and pharmaceutical industries as
an alternative to in vivo models to determine toxic potential,
toxic potency, and for hazard identification of chemicals Skin
models have been developed for toxicology assessment as an
alternative to in vivo models. These skin models generally follow
methods detailed in Organization for Economic Co-operation and
Development (OECD) test guidelines (TG) 439 and 431 for
reconstructed human epidermis skin irritation and corrosion,
respectively Skin irritation refers to the production of reversible
damage to the skin following the application of a test chemical for
up to 4 hours [as defined by the United Nations (UN) Globally
Harmonized System of Classification and Labelling of Chemicals
(GHS)]. Skin corrosion refers to the production of irreversible
damage to the skin manifested as visible necrosis through the
epidermis and into the dermis, following the application of a test
material (UN GHS). Provided herein, are non-limiting examples of
usage for engineered 3D skin tissue as an in vitro system to model
toxicology. In addition to prediction and classification of test
substances as irritating or corrosive, provided herein are in vitro
human skin models that are sufficiently complex to mimic
morphology, cell reactivity and barrier function of native skin can
be used to predict phototoxicity, genotoxicity, sensitization,
penetration, absorption, adsorption, and to model transdermal drug
delivery.
[0072] Fabricating skin with the bioprinting platform disclosed
herein compared to current skin models and natural skin is that the
process is automated. This allows for greater reproducibility and
scalability. For example, it is possible to miniaturize the tissue
geometry in order to print bio-ink into well plate formats such as
6, 12, 24, 48, 96, 384 or 1536-well plates for use in screening
applications including high-throughput screening applications.
Another major advantage of an automated platform is that it can be
utilized to administer substances for toxicity testing in addition
to bioprinting tissue. Current testing in skin models is limited by
the manual approaches necessary both to fabricate the tissue and to
apply a test material to that tissue, limiting the application to
topical administration. The flexibility of the printing platform
allows for a variety of methods for application, deposition, and
incorporation into tissues not possible with a manual approach. For
example, test articles could be sprayed in a fine mist using the
inkjet technology, or injected into the dermal layer utilizing the
continuous deposition module. A third major advantage of
bioprinting in a skin toxicology model is the time frame in which a
layered structure can be generated and tested. Current 3D skin
models often require a minimum of 4 weeks to obtain a layered
skin-like structure. Bioprinting approaches can overlay sheets of
cells simultaneously or with a delay to create dermal and epidermal
layers which can then be allowed to mature and differentiate for a
defined period of time. The bioprinting platform allows for
longitudinal studies not possible with manual approaches because
test substances can be exposed to or incorporated into tissues
during printing or administered to mature tissues at later time
points. The skin tissue testing method presented in this disclosure
allows for application and analysis of a potentially toxic test
substance to a tissue exhibiting layered architecture.
[0073] In some embodiments, the three-dimensional, engineered,
biological skin tissues described herein include one or more
compartments or cellular layers. In some embodiments, the
engineered skin tissues consist essentially of a dermal layer or
dermal compartment. In other embodiments, the engineered skin
tissues consist essentially of an epidermal layer or epidermal
compartment. In yet other embodiments, the engineered skin tissues
are full-thickness skin tissues compared a dermal layer or dermal
compartment and an epidermal layer or epidermal compartment. In yet
other embodiments, the engineered skin tissues are full-thickness
skin tissues consisting essentially of a dermal layer or dermal
compartment and an epidermal layer or epidermal compartment. In
further embodiments, the epidermal layer or compartment is
stratified. In some embodiments, the engineered skin tissues
comprise a hypodermal layer or compartment. In some embodiments,
the engineered skin tissues comprise a basal layer or compartment.
In yet another embodiment the skin tissue consists essentially of
an epidermal layer, a dermal layer and a hypodermal layer.
[0074] In some embodiments, the tissues, arrays, and methods
described herein include one or more adherent cell types, or use of
the same. Many cell types are suitable for inclusion in the
engineered skin tissues. By way of example, in some embodiments,
the engineered skin tissues include dermal fibroblasts and
keratinocytes. By way of further example, in some embodiments, the
engineered skin tissues include melanocytes. By way of further
example, in some embodiments, the engineered skin tissues include
secretory cells and/or immune cells. In a particular embodiment,
the engineered skin tissues include cancer cells. In a particular
embodiment, the engineered skin tissues include cells derived from
induced pluripotent stem (iPS) cells or embryonic stem (ES) cells.
In some embodiments, the cells are human cells. In some
embodiments, the cells are primary cells. In some embodiments, the
cells are primary human cells.
[0075] In some embodiments, the cells are bioprinted. In further
embodiments, the bioprinted cells are cohered to form the
engineered skin tissues. In still further embodiments, the
engineered skin tissues are free or substantially free of
pre-formed scaffold at the time of fabrication or the time of use.
In some cases, bioprinting allows fabrication of tissues that mimic
the appropriate cellularity of native tissue. In some embodiments,
the cells are bioprinted by an extrusion method. In some
embodiments, the cells are bioprinted by an aerosol spray
method.
[0076] In some embodiments, the three-dimensional, engineered skin
tissues described herein are distinguished from tissues fabricated
by prior technologies by virtue of the fact that they are
three-dimensional, free of pre-formed scaffolds, consist
essentially of cells, and/or have a high cell density (e.g.,
greater than 30% cellular, greater than 40% cellular, greater than
50% cellular, greater than 60% cellular, greater than 70% cellular,
greater than 80% cellular, or greater than 90% cellular).
Distinguished from Native Tissue
[0077] In some embodiments, the three-dimensional, engineered skin
tissues described herein are distinguished from native (e.g.,
non-engineered or fabricated) tissues by virtue of the fact that
they are non-innervated (e.g., substantially free of nervous
tissue), substantially free of mature vasculature, and/or
substantially free of blood components. In certain embodiments, the
tissues lack perfusable vasculature. For example, in various
embodiments, the three-dimensional, engineered skin tissues are
free of plasma, red blood cells, platelets, and the like and/or
endogenously-generated plasma, red blood cells, platelets, and the
like. In certain embodiments, the tissues lack hemoglobin. In some
embodiments, the tissues lack innervation or neurons. In some
embodiments, the tissue lack cells expressing neuronal markers such
as any of: Beta III tubulin, MAP2, NeuN and neuron specific
enolase. In certain embodiments, the tissues are free of
lymphatics. In certain embodiments, the tissues are free of immune
cells. In certain embodiments, the tissues are free of Langerhans
cells. In certain embodiments, the tissues are free of T-cells. In
certain embodiments, the cells are substantially free of any of the
immune cells marked by the following proteins: CD11c, DC-SIGN,
CD11b, CD4, CD8, CD28, CD3, CD19, CD80, and/or CD86.
[0078] The tissues of the current disclosure are marked by extended
viability in culture. Traditional tissue explants exhibit low
viability in in vitro culture. In certain embodiments, the
three-dimensional, engineered skin tissues described herein are
viable after 7 days or more in culture. In certain embodiments, the
three-dimensional, engineered skin tissues described herein are
viable after 10 days or more in culture. In certain embodiments,
the three-dimensional, engineered skin tissues described herein are
viable after 14 days or more in culture. In certain embodiments,
the three-dimensional, engineered skin tissues described herein are
viable after 21 days or more in culture. In certain embodiments,
bioprinted tissues possess a higher basal metabolic rate than
tissues directly ex vivo. In certain embodiments, bioprinted
tissues possess a higher proliferative rate than tissues directly
ex vivo or tissues in vivo.
[0079] In certain embodiments, greater than 50% of the cells of the
three-dimensional, engineered skin tissue are live after 7 days
post bioprinting. In certain embodiments, greater than 70% of the
cells of the three-dimensional, engineered skin tissue are live
after 7 days post bioprinting. In certain embodiments, greater than
90% of the cells of the three-dimensional, engineered skin tissue
are live after 7 days post bioprinting. In certain embodiments,
greater than 50% of the cells of the three-dimensional, engineered
skin tissue are live after 14 days post bioprinting. In certain
embodiments, greater than 70% of the cells of the
three-dimensional, engineered skin tissue are live after 14 days
post bioprinting. In certain embodiments, greater than 90% of the
cells of the three-dimensional, engineered skin tissue are live
after 14 days post bioprinting. In certain embodiments, greater
than 50% of the cells of the three-dimensional, engineered skin
tissue are live after 21 days post bioprinting. In certain
embodiments, greater than 70% of the cells of the
three-dimensional, engineered skin tissue are live after 21 days
post bioprinting. In certain embodiments, greater than 90% of the
cells of the three-dimensional, engineered skin tissue are live
after 21 days post bioprinting.
[0080] One advantage of the tissues fabricated by the methods of
this disclosure is the ability to form novel and advantageous
chimeras. In some embodiments, the engineered skin tissues are
species chimeras, wherein at least one cell or cell-type of the
tissue is from a different mammalian species than another cell or
cell-type of the tissue. For example, the dermal bio-ink contains a
cell of mouse, rat, or primate origin and the epidermal bio-ink
contains a cell of human origin. In some embodiments, the
engineered skin tissues are genetic chimeras, wherein at least one
cell or cell-type is from a different genetic background (e.g.,
different genotype, etc.) than the genetic background of any other
cell or cell-type of the tissue. For example, the dermal
fibroblasts of the dermal bio-ink may be from a certain donor and
the keratinocytes or melanocytes of the epidermal bio-ink may be
from a different donor, creating a genetic chimera. In some
embodiments, the engineered skin tissues are chimeras of other
types. For example, the dermal bio-ink may comprise a transformed
dermal fibroblast, and the epidermal bio-ink may comprise a primary
untransformed keratinocyte or melanocyte. In certain embodiments,
the dermal bio-ink may contain fibroblasts of non-dermal origin. In
certain embodiments, the hypodermal bio-ink may contain endothelial
cells of non-dermal origin.
[0081] FIG. 1 illustrates a cross-section of native skin tissue. As
illustrated in FIG. 1, native human skin includes an epidermal
layer (keratinocytes) 1 (further comprising a stratum corneum 1a,
stratum granulosum 1b, stratum spinosum 1c, and stratum basale 1d),
a dermal layer 2 (further comprising a papillary dermal layer 2a
and peticular dermal layer 2b), a subcutaneous fatty tissue layer
3, a connective tissue layer 4, and a muscle layer 5.
[0082] Further, as illustrated in FIG. 1, native human skin
includes keratinocytes 6, melanocytes 7, fibroblasts 8 (including
papillary dermal fibroblasts 8a and reticular dermal fibroblasts
8b), Merkel cells 9, Langerhans cells 10, macrophages 11, stem
cells 12, endothelial cells 13, epithelial cells 14, adipocytes 15,
muscle cells 16, and sensory neurons 17.
[0083] Further, as illustrated in FIG. 1, native human skin
includes organoids 18, blood vessels 19, lymphatic vessels 20, hair
follicles 21, sebaceous glands 22, sweat glands 23, pores 24,
muscle 28, arrector pili muscle 29, Meissner's corpuscle 30,
Pacinian corpuscle 31, Ruffini corpuscle 32, loose connective
tissue 33, dense connective tissue 34, and basement membrane
35.
[0084] Further, as illustrated in FIG. 1, native human skin, in
some cases, includes basal cell carcinoma 25, squamous cell
carcinoma 26, and melanoma 27.
[0085] In some embodiments, one or more components of the
engineered skin tissue described herein are bioprinted, which
comprises an additive fabrication process. Therefore, in such
embodiments, through the methods of fabrication, the fabricator
exerts significant control over the composition of the resulting
engineered skin tissues described herein. As such, the engineered
skin tissues described herein optionally comprise any of the
layers, structures, compartments, and/or cells of native tissue.
Conversely, the engineered skin tissues described herein optionally
lack any of the layers, structures, compartments, and/or cells of
native tissue.
[0086] Referring to FIG. 1, in some embodiments, an engineered skin
tissue described herein does not comprise (e.g., lacks) layers or
compartments selected from any of the following: a stratum corneum
1a, stratum granulosum 1b, stratum spinosum 1c, and stratum basale
1d; a papillary dermal layer 2a and peticular dermal layer 2b, a
subcutaneous fatty tissue layer 3, a connective tissue layer 4, and
a muscle layer 5. Further, in certain embodiments, an engineered
skin tissue described herein does not comprise cells selected from:
keratinocytes 6, melanocytes 7, fibroblasts 8 (including papillary
dermal fibroblasts 8a and reticular dermal fibroblasts 8b), Merkel
cells 9, Langerhans cells 10, macrophages 11, stem cells 12,
endothelial cells 13, epithelial cells 14, adipocytes 15, muscle
cells 16, and sensory neurons 17. Further, in certain embodiments,
an engineered skin tissue described herein does not comprise
structures selected from: organoids 18, blood vessels 19, lymphatic
vessels 20, hair follicles 21, sebaceous glands 22, sweat glands
23, pores 24, basal cell carcinoma 25, squamous cell carcinoma 26,
melanoma 27, muscle 28, arrector pili muscle 29, Meissner's
corpuscle 30, Pacinian corpuscle 31, Ruffini corpuscle 32, loose
connective tissue 33, dense connective tissue 34, and basement
membrane 35.
[0087] Referring to FIG. 2, normal human skin tissue includes an
epidermal layer stratified into basal, spinous, granular, and
cornified layers, atop a basement membrane, which in turn rests
upon a layer of connective tissue. A variety of biomarkers are
optionally utilized to visualize these layers.
Bio-Ink
[0088] The tissues, arrays, and methods described herein involve
bio-ink formulations and bioprinting methods to create 3D skin
tissue structures. In certain embodiments, the bio-ink is dermal,
epidermal, hypodermal, basal non-cellular bio-ink, or any
combination thereof.
[0089] The tissues, arrays, and methods described herein involve
bio-ink formulations and bioprinting methods to create 3D skin
tissue structures containing compositions of keratinocytes,
fibroblasts, and/or melanocytes or endothelial cells. The printing
methods utilize bio-ink to create geometries which produce layers
or compartments to mimic native skin. Bioprinted tissues optionally
model the dermis, epidermis, or a combination of both. In various
embodiments, the bio-ink contains a cellular mixture of some
proportion of keratinocytes, fibroblasts, and/or melanocytes and
optionally contains a biomaterial support. In other embodiments,
the bio-ink contains some portion of keratinocytes, melanocytes,
fibroblasts (including papillary dermal fibroblasts and reticular
dermal fibroblasts), Merkel cells, Langerhans cells, macrophages,
stem cells, endothelial cells, epithelial cells, adipocytes, muscle
cells, and sensory neuronal cells and optionally contains a
biomaterial support. In various embodiments, the printing methods
utilize a variety of printing surfaces with a variety of pore sizes
that are optionally coated with matrix support material such as
collagen. In some embodiments, hydrogels are optionally added to
support biomaterials or constitute space-saving regions in which
there are no cells. In various embodiments, the skin tissue
comprises epidermal bio-inks, dermal bio-inks or both. In certain
embodiments, bio-inks consist essentially of a certain cell
type.
[0090] Consisting essentially means that the specified cell type is
the only cell type present, but the bio-ink may contain other
non-cellular material including but not limited to extrusion
compounds, hydrogels, extracellular matrix components, nutritive
and media components, inorganic and organic salts, acids and bases,
buffer compounds and other non-cellular components that promote
cell survival, adhesion, growth, or facilitate printing.
[0091] 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, gelatin, 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.
[0092] Suitable hydrogels include those derived from collagen,
hyaluronate, hyaluronan, fibrin, alginate, agarose, chitosan,
chitin, cellulose, pectin, starch, polysaccharides,
fibrinogen/thrombin, fibrillin, elastin, gum, cellulose, agar,
gluten, casein, albumin, vitronectin, tenascin, entactin/nidogen,
glycoproteins, glycosaminoglycans (GAGs) and proteoglycans which
may contain for example chrondroitin sulfate, fibronectin, keratin
sulfate, laminin, heparan sulfate proteoglycan, decorin, aggrecan,
perlecan or any combination 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.
[0093] In certain embodiments, the bio-ink is a viscous liquid. In
certain embodiments, the bio-ink is a semi-solid. In certain
embodiments, the bio-ink is a solid. In certain embodiments, the
bio-ink is a semi-solid or a solid. In certain embodiments, the
viscosity of the bio-ink is greater than 100 centipoise. In certain
embodiments, the viscosity of the bio-ink is greater than 200
centipoise. In certain embodiments, the viscosity of the bio-ink is
greater than 500 centipoise. In certain embodiments, the viscosity
of the bio-ink is greater than 1,000 centipoise. In certain
embodiments, the viscosity of the bio-ink is greater than 2,000
centipoise. In certain embodiments, the viscosity of the bio-ink is
greater than 5,000 centipoise. In certain embodiments, the
viscosity of the bio-ink is greater than 10,000 centipoise. In
certain embodiments, the viscosity of the bio-ink is greater than
20,000 centipoise. In certain embodiments, the viscosity of the
bio-ink is greater than 50,000 centipoise. In certain embodiments,
the viscosity of the bio-ink is greater than 100,000 centipoise. In
certain embodiments, the viscosity of the bio-ink is less than 100
centipoise. In certain embodiments, the viscosity of the bio-ink is
less than 200 centipoise. In certain embodiments, the viscosity of
the bio-ink is less than 500 centipoise. In certain embodiments,
the viscosity of the bio-ink is less than 1,000 centipoise. In
certain embodiments, the viscosity of the bio-ink is less than
2,000 centipoise. In certain embodiments, the viscosity of the
bio-ink is less than 5,000 centipoise. In certain embodiments, the
viscosity of the bio-ink is less than 10,000 centipoise. In certain
embodiments, the viscosity of the bio-ink is less than 20,000
centipoise. In certain embodiments, the viscosity of the bio-ink is
less than 50,000 centipoise. In certain embodiments, the viscosity
of the bio-ink is less than 100,000 centipoise.
Dermal Bio-Ink
[0094] In some embodiments, the three-dimensional, engineered skin
tissues comprise a dermal bio-ink. In certain embodiments, the
dermal bio-ink comprises fibroblasts. In certain embodiments, the
dermal bio-ink comprises dermal fibroblasts. In certain
embodiments, the dermal bio-ink comprises human dermal fibroblasts.
In certain embodiments, the dermal bio-ink comprises primary human
dermal fibroblasts. In certain embodiments, the dermal bio-ink
comprises non-dermal fibroblasts. In certain embodiments, the
dermal bio-ink consists essentially of fibroblasts. In certain
embodiments, the dermal bio-ink consists essentially of dermal
fibroblasts. In certain embodiments, the dermal bio-ink consists
essentially of human dermal fibroblasts. In certain embodiments,
the dermal bio-ink consists essentially of primary human dermal
fibroblasts. In certain embodiments, the dermal bio-ink consists
essentially of non-dermal fibroblasts.
[0095] In certain embodiments, the dermal bio-ink comprises greater
than 50% live cells by volume. In certain embodiments, the dermal
bio-ink comprises greater than 60% live cells by volume. In certain
embodiments, the dermal bio-ink comprises greater than 70% live
cells by volume. In certain embodiments, the dermal bio-ink
comprises greater than 80% live cells by volume. In certain
embodiments, the dermal bio-ink comprises greater than 90% live
cells by volume. In certain embodiments, the dermal bio-ink
comprises greater than 95% live cells by volume.
[0096] In certain embodiments, the dermal bio-ink can be applied as
a layer or an individual compartment by an aerosol spray method. In
certain embodiments, the dermal bio-ink comprises between 0.05
million and 50 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises between 0.1 million and
50 million cells per milliliter. In certain embodiments, the dermal
bio-ink comprises between 0.1 million and 40 million cells per
milliliter. In certain embodiments, the dermal bio-ink comprises
between 0.1 million and 30 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises between 0.5 million and
50 million cells per milliliter. In certain embodiments, the dermal
bio-ink comprises between 0.5 million and 40 million cells per
milliliter. In certain embodiments, the dermal bio-ink comprises
between 0.5 million and 30 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises between 1 million and 50
million cells per milliliter. In certain embodiments, the dermal
bio-ink comprises between 1 million and 40 million cells per
milliliter. In certain embodiments, the dermal bio-ink comprises
between 1 million and 30 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises between 10 million and 50
million cells per milliliter. In certain embodiments, the dermal
bio-ink comprises between 10 million and 40 million cells per
milliliter. In certain embodiments, the dermal bio-ink comprises
between 10 million and 30 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises between 2 million and 50
million cells per milliliter. In certain embodiments, the dermal
bio-ink comprises between 3 million and 50 million cells per
milliliter. In certain embodiments, the dermal bio-ink comprises
between 4 million and 50 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises between 5 million and 50
million cells per milliliter. In certain embodiments, the dermal
bio-ink comprises less than 50 million cells per milliliter. In
certain embodiments, the dermal bio-ink comprises less than 40
million cells per milliliter. In certain embodiments, the dermal
bio-ink comprises less than 30 million cells per milliliter. In
certain embodiments, the dermal bio-ink comprises less than 25
million cells per milliliter. In certain embodiments, the dermal
bio-ink comprises less than 10 million cells per milliliter.
[0097] In certain embodiments, the dermal bio-ink can be applied as
a layer or an individual compartment by an extrusion method. In
certain embodiments, the dermal bio-ink comprises between 5.0
million and 500 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises between 5.0 million and
400 million cells per milliliter. In certain embodiments, the
dermal bio-ink comprises between 5.0 million and 300 million cells
per milliliter. In certain embodiments, the dermal bio-ink
comprises between 10 million and 500 million cells per milliliter.
In certain embodiments, the dermal bio-ink comprises between 10
million and 400 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises between 10 million and
300 million cells per milliliter. In certain embodiments, the
dermal bio-ink comprises between 10 million and 200 million cells
per milliliter. In certain embodiments, the dermal bio-ink
comprises between 10 million and 100 million cells per milliliter.
In certain embodiments, the dermal bio-ink comprises between 10
million and 50 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises between 100 million and
500 million cells per milliliter. In certain embodiments, the
dermal bio-ink comprises between 100 million and 400 million cells
per milliliter. In certain embodiments, the dermal bio-ink
comprises between 100 million and 300 million cells per milliliter.
In certain embodiments, the dermal bio-ink comprises between 100
million and 200 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises between 25 million and
200 million cells per milliliter. In certain embodiments, the
dermal bio-ink comprises greater than 50 million cells per
milliliter. In certain embodiments, the dermal bio-ink comprises
greater than 100 million cells per milliliter. In certain
embodiments, the dermal bio-ink comprises greater than 200 million
cells per milliliter.
Epidermal Bio-Ink
[0098] In some embodiments, the three-dimensional, engineered skin
tissues comprise an epidermal bio-ink. In certain embodiments, the
epidermal bio-ink comprises keratinocytes. In certain embodiments,
the epidermal bio-ink comprises melanocytes. In certain
embodiments, the epidermal bio-ink comprises keratinocytes and
melanocytes. In certain embodiments, the epidermal bio-ink
comprises primary keratinocytes. In certain embodiments, the
epidermal bio-ink comprises primary melanocytes. In certain
embodiments, the epidermal bio-ink comprises primary keratinocytes
and primary melanocytes. In some embodiments, the
three-dimensional, engineered skin tissue consists essentially of
an epidermal bio-ink. In certain embodiments, the epidermal bio-ink
consists essentially of keratinocytes. In certain embodiments, the
epidermal bio-ink consists essentially of melanocytes. In certain
embodiments, the epidermal bio-ink consists essentially of
keratinocytes and melanocytes. In certain embodiments, the
epidermal bio-ink consists essentially of primary keratinocytes. In
certain embodiments, the epidermal bio-ink consists essentially of
primary melanocytes. In certain embodiments, the epidermal bio-ink
comprises consists essentially of keratinocytes and primary
melanocytes. In certain embodiments, the epidermal bio-ink
comprises human keratinocytes. In certain embodiments, the
epidermal bio-ink comprises human melanocytes. In certain
embodiments, the epidermal bio-ink comprises human keratinocytes
and human melanocytes. In certain embodiments, the epidermal
bio-ink comprises human primary keratinocytes. In certain
embodiments, the epidermal bio-ink comprises human primary
melanocytes. In certain embodiments, the epidermal bio-ink
comprises human primary keratinocytes and human primary
melanocytes. In certain embodiments, the epidermal bio-ink consists
essentially of human keratinocytes. In certain embodiments, the
epidermal bio-ink consists essentially of human melanocytes. In
certain embodiments, the epidermal bio-ink consists essentially of
human keratinocytes and human melanocytes. In certain embodiments,
the epidermal bio-ink consists essentially of human primary
keratinocytes. In certain embodiments, the epidermal bio-ink
consists essentially of human primary melanocytes. In certain
embodiments, the epidermal bio-ink comprises consists essentially
of human keratinocytes and human primary melanocytes.
[0099] In certain embodiments, the epidermal bio-ink comprises,
consists essentially of, or consists of keratinocytes and
melanocytes, primary or non-primary, at specified ratios. In
certain embodiments, the ratio of keratinocytes to melanocytes is
from about 75:25 to about 99:1. In certain embodiments, the ratio
of keratinocytes to melanocytes is from about 80:20 to about 99:1.
In certain embodiments, the ratio of keratinocytes to melanocytes
is from about 85:15 to about 99:1. In certain embodiments, the
ratio of keratinocytes to melanocytes is from about 88:12 to about
99:1. In certain embodiments, the ratio of keratinocytes to
melanocytes is from about 89:11 to about 99:1. In certain
embodiments, the ratio of keratinocytes to melanocytes is from
about 90:10 to about 99:1. In certain embodiments, the ratio of
keratinocytes to melanocytes is from about 91:9 to about 99:1. In
certain embodiments, the ratio of keratinocytes to melanocytes is
from about 94:6 to about 99:1. In certain embodiments, the ratio of
keratinocytes to melanocytes is from about 95:5 to about 99:1. In
certain embodiments, the ratio of keratinocytes to melanocytes is
from about 96:4 to about 99:1.
[0100] In certain embodiments, the epidermal bio-ink comprises
greater than 50% live cells by volume. In certain embodiments, the
epidermal bio-ink comprises greater than 60% live cells by volume.
In certain embodiments, the epidermal bio-ink comprises greater
than 70% live cells by volume. In certain embodiments, the
epidermal bio-ink comprises greater than 80% live cells by volume.
In certain embodiments, the epidermal bio-ink comprises greater
than 90% live cells by volume. In certain embodiments, the
epidermal bio-ink comprises greater than 95% live cells by
volume.
[0101] In certain embodiments, the epidermal bio-ink can be applied
as a layer or an individual compartment by an aerosol spray method.
In certain embodiments, the epidermal bio-ink comprises between
0.05 million and 50 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises between 0.1 million
and 50 million cells per milliliter. In certain embodiments, the
epidermal bio-ink comprises between 0.1 million and 40 million
cells per milliliter. In certain embodiments, the epidermal bio-ink
comprises between 0.1 million and 30 million cells per milliliter.
In certain embodiments, the epidermal bio-ink comprises between 0.5
million and 50 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises between 0.5 million
and 40 million cells per milliliter. In certain embodiments, the
epidermal bio-ink comprises between 0.5 million and 30 million
cells per milliliter. In certain embodiments, the epidermal bio-ink
comprises between 1 million and 50 million cells per milliliter. In
certain embodiments, the epidermal bio-ink comprises between 1
million and 40 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises between 1 million and
30 million cells per milliliter. In certain embodiments, the
epidermal bio-ink comprises between 10 million and 50 million cells
per milliliter. In certain embodiments, the epidermal bio-ink
comprises between 10 million and 40 million cells per milliliter.
In certain embodiments, the epidermal bio-ink comprises between 10
million and 30 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises between 2 million and
50 million cells per milliliter. In certain embodiments, the
epidermal bio-ink comprises between 3 million and 50 million cells
per milliliter. In certain embodiments, the epidermal bio-ink
comprises between 4 million and 50 million cells per milliliter. In
certain embodiments, the epidermal bio-ink comprises between 5
million and 50 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises less than 50 million
cells per milliliter. In certain embodiments, the epidermal bio-ink
comprises less than 40 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises less than 30 million
cells per milliliter. In certain embodiments, the epidermal bio-ink
comprises less than 25 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises less than 10 million
cells per milliliter.
[0102] In certain embodiments, the epidermal bio-ink can be applied
as a layer or an individual compartment by an extrusion method. In
certain embodiments, the epidermal bio-ink comprises between 5.0
million and 500 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises between 5.0 million
and 400 million cells per milliliter. In certain embodiments, the
epidermal bio-ink comprises between 5.0 million and 300 million
cells per milliliter. In certain embodiments, the epidermal bio-ink
comprises between 10 million and 500 million cells per milliliter.
In certain embodiments, the epidermal bio-ink comprises between 10
million and 400 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises between 10 million and
300 million cells per milliliter. In certain embodiments, the
epidermal bio-ink comprises between 10 million and 200 million
cells per milliliter. In certain embodiments, the epidermal bio-ink
comprises between 10 million and 100 million cells per milliliter.
In certain embodiments, the epidermal bio-ink comprises between 10
million and 50 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises between 100 million
and 500 million cells per milliliter. In certain embodiments, the
epidermal bio-ink comprises between 100 million and 400 million
cells per milliliter. In certain embodiments, the epidermal bio-ink
comprises between 100 million and 300 million cells per milliliter.
In certain embodiments, the epidermal bio-ink comprises between 100
million and 200 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises between 25 million and
200 million cells per milliliter. In certain embodiments, the
epidermal bio-ink comprises greater than 50 million cells per
milliliter. In certain embodiments, the epidermal bio-ink comprises
greater than 100 million cells per milliliter. In certain
embodiments, the epidermal bio-ink comprises greater than 200
million cells per milliliter.
Hypodermal Bio-Ink
[0103] In certain embodiments, the hypodermal bio-ink comprises
endothelial cells. In certain embodiments, the hypodermal bio-ink
comprises fibroblasts. In certain embodiments, the hypodermal
bio-ink comprises endothelial cells and fibroblasts. In certain
embodiments, the hypodermal bio-ink comprises human endothelial
cells. In certain embodiments, the hypodermal bio-ink comprises
human fibroblasts. In certain embodiments, the hypodermal bio-ink
comprises human endothelial cells and human fibroblasts. In certain
embodiments, the hypodermal bio-ink comprises human primary
endothelial cells. In certain embodiments, the hypodermal bio-ink
comprises human primary fibroblasts. In certain embodiments, the
hypodermal bio-ink comprises human primary endothelial cells and
human primary fibroblasts. In certain embodiments, the hypodermal
bio-ink consists essentially of endothelial cells. In certain
embodiments, the hypodermal bio-ink consists essentially of
fibroblasts. In certain embodiments, the hypodermal bio-ink
consists essentially of endothelial cells and fibroblasts. In
certain embodiments, the hypodermal bio-ink consists essentially of
human endothelial cells. In certain embodiments, the hypodermal
bio-ink consists essentially of human fibroblasts. In certain
embodiments, the hypodermal bio-ink consists essentially of human
endothelial cells and human fibroblasts. In certain embodiments,
the hypodermal bio-ink consists essentially of human primary
endothelial cells. In certain embodiments, the hypodermal bio-ink
consists essentially of human primary fibroblasts. In certain
embodiments, the hypodermal bio-ink consists essentially of human
primary endothelial cells and human primary fibroblasts.
[0104] In certain embodiments, the hypodermal bio-ink comprises
greater than 50% live cells by volume. In certain embodiments, the
hypodermal bio-ink comprises greater than 60% live cells by volume.
In certain embodiments, the hypodermal bio-ink comprises greater
than 70% live cells by volume. In certain embodiments, the
hypodermal bio-ink comprises greater than 80% live cells by volume.
In certain embodiments, the hypodermal bio-ink comprises greater
than 90% live cells by volume. In certain embodiments, the
hypodermal bio-ink comprises greater than 95% live cells by
volume.
[0105] In certain embodiments, the hypodermal bio-ink can be
applied as a layer or an individual compartment by an aerosol spray
method. In certain embodiments, the hypodermal bio-ink comprises
between 0.05 million and 50 million cells per milliliter. In
certain embodiments, the hypodermal bio-ink comprises between 0.1
million and 50 million cells per milliliter. In certain
embodiments, the hypodermal bio-ink comprises between 0.1 million
and 40 million cells per milliliter. In certain embodiments, the
hypodermal bio-ink comprises between 0.1 million and 30 million
cells per milliliter. In certain embodiments, the hypodermal
bio-ink comprises between 0.5 million and 50 million cells per
milliliter. In certain embodiments, the hypodermal bio-ink
comprises between 0.5 million and 40 million cells per milliliter.
In certain embodiments, the hypodermal bio-ink comprises between
0.5 million and 30 million cells per milliliter. In certain
embodiments, the hypodermal bio-ink comprises between 1 million and
50 million cells per milliliter. In certain embodiments, the
hypodermal bio-ink comprises between 1 million and 40 million cells
per milliliter. In certain embodiments, the hypodermal bio-ink
comprises between 1 million and 30 million cells per milliliter. In
certain embodiments, the hypodermal bio-ink comprises between 10
million and 50 million cells per milliliter. In certain
embodiments, the hypodermal bio-ink comprises between 10 million
and 40 million cells per milliliter. In certain embodiments, the
hypodermal bio-ink comprises between 10 million and 30 million
cells per milliliter. In certain embodiments, the hypodermal
bio-ink comprises between 2 million and 50 million cells per
milliliter. In certain embodiments, the hypodermal bio-ink
comprises between 3 million and 50 million cells per milliliter. In
certain embodiments, the hypodermal bio-ink comprises between 4
million and 50 million cells per milliliter. In certain
embodiments, the hypodermal bio-ink comprises between 5 million and
50 million cells per milliliter. In certain embodiments, the
hypodermal bio-ink comprises less than 50 million cells per
milliliter. In certain embodiments, the hypodermal bio-ink
comprises less than 40 million cells per milliliter. In certain
embodiments, the hypodermal bio-ink comprises less than 30 million
cells per milliliter. In certain embodiments, the hypodermal
bio-ink comprises less than 25 million cells per milliliter. In
certain embodiments, the hypodermal bio-ink comprises less than 10
million cells per milliliter.
[0106] In certain embodiments, the hypodermal bio-ink can be
applied as a layer or an individual compartment by an extrusion
method. In certain embodiments, the hypodermal bio-ink comprises
between 5.0 million and 500 million cells per milliliter. In
certain embodiments, the hypodermal bio-ink comprises between 5.0
million and 400 million cells per milliliter. In certain
embodiments, the hypodermal bio-ink comprises between 5.0 million
and 300 million cells per milliliter. In certain embodiments, the
hypodermal bio-ink comprises between 10 million and 500 million
cells per milliliter. In certain embodiments, the hypodermal
bio-ink comprises between 10 million and 400 million cells per
milliliter. In certain embodiments, the hypodermal bio-ink
comprises between 10 million and 300 million cells per milliliter.
In certain embodiments, the hypodermal bio-ink comprises between 10
million and 200 million cells per milliliter. In certain
embodiments, the hypodermal bio-ink comprises between 10 million
and 100 million cells per milliliter. In certain embodiments, the
hypodermal bio-ink comprises between 10 million and 50 million
cells per milliliter. In certain embodiments, the hypodermal
bio-ink comprises between 100 million and 500 million cells per
milliliter. In certain embodiments, the hypodermal bio-ink
comprises between 100 million and 400 million cells per milliliter.
In certain embodiments, the hypodermal bio-ink comprises between
100 million and 300 million cells per milliliter. In certain
embodiments, the hypodermal bio-ink comprises between 100 million
and 200 million cells per milliliter. In certain embodiments, the
hypodermal bio-ink comprises between 25 million and 200 million
cells per milliliter. In certain embodiments, the hypodermal
bio-ink comprises greater than 50 million cells per milliliter. In
certain embodiments, the hypodermal bio-ink comprises greater than
100 million cells per milliliter. In certain embodiments, the
hypodermal bio-ink comprises greater than 200 million cells per
milliliter.
Non-Cellular Bio-Inks
[0107] In some embodiments, the three-dimensional, engineered skin
tissues comprise a non-cellular bio-ink. In some embodiments, the
non-cellular bio-ink comprises extracellular matrix proteins or
peptides such as collagen or fibrinogen, hyaluronate, hyaluronan,
fibrin, alginate, agarose, chitosan, chitin, cellulose, pectin,
starch, polysaccharides, fibrinogen/thrombin, fibrillin, elastin,
gum, cellulose, agar, gluten, casein, albumin, vitronectin,
tenascin, entactin/nidogen, glycoproteins, glycosaminoglycans
(GAGs) and proteoglycans which may contain for example chrondroitin
sulfate, fibronectin, keratin sulfate, laminin, heparan sulfate
proteoglycan, decorin, aggrecan, perlecan or any 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. In some embodiments, the
non-cellular bio-ink comprises hydrogels or other support
materials, cushion materials or confinement materials. In some
embodiments, the non-cellular bio-ink does not comprise inorganic
or synthetic polymer. In some embodiments, the non-cellular bio-ink
does not comprise dead-cell debris.
[0108] In some embodiments, the engineered tissues, arrays, and
methods described herein incorporate continuous deposition printing
into a 3D skin model. Continuous deposition is optionally utilized
to produce single or multiple layers mimicking the dermis and/or
epidermis. In one embodiment, a bio-ink comprised of fibroblasts is
printed to produce a tissue mimicking the dermis. In another
embodiment, bio-ink comprised of keratinocytes or a mixture of
keratinocytes and melanocytes is printed to produce a tissue to
mimic the epidermis. A third embodiment combines bio-inks to
simultaneously deposit the epidermal bio-ink on top of the dermal
bio-ink. Continuous deposition printing provides an advantage to
current 3D skin models in that it enables cells to be placed within
a precise geometry and enables the use of multiple bio-ink
formulations including, but not limited to, Novogel.RTM. 2.0,
Novogel.RTM. 3.0, and cell paste. Continuous deposition allows
optional incorporation of various biomaterials into the
Novogel.RTM. formulation and various printing surfaces to promote
extracellular matrix production and differentiation.
Tissue Architectures
[0109] The three-dimensional, engineered skin tissues, arrays, and
methods described herein, allow for the generation of multi
layered, compartmentalized engineered constructs. The layers are
formed by bio-inks disposed upon a surface. The bio-inks then
cohere to form a single tissue with a plurality of layers and/or
discrete compartments. In certain embodiments, the tissue layers or
compartments are architecturally distinct. In certain embodiments,
the skin tissues comprise an epidermal layer. In certain
embodiments, the skin tissues comprise a dermal layer. In certain
embodiments, the skin tissues comprise a hypodermal layer. In
certain embodiments, the skin tissues comprise an epidermal layer
disposed on top of a dermal layer. In certain embodiments, the skin
tissues comprise an epidermal layer disposed on top of a dermal
layer, disposed on top of a hypodermal layer. In certain
embodiments, the skin tissues comprise an epidermal layer disposed
on top of non-cellular layer, disposed on top of a dermal layer. In
certain embodiments, the skin tissues comprise an epidermal layer
disposed on top of a non-cellular layer disposed on top of a dermal
layer, deposed on top of a hypodermal layer. In certain
embodiments, the tissue layers are architecturally distinct. In
certain embodiments, the skin tissues consist of an epidermal
layer. In certain embodiments, the skin tissues consist of a dermal
layer. In certain embodiments, the skin tissues consist of a
hypodermal layer. In certain embodiments, the skin tissues consist
of an epidermal layer disposed on top of a dermal layer. In certain
embodiments, the skin tissues consist of an epidermal layer
disposed on top of a dermal layer, disposed on top of a hypodermal
layer. In certain embodiments, the skin tissues consist of an
epidermal layer disposed on top of non-cellular layer, disposed on
top of a dermal layer. In certain embodiments, the skin tissues
consist of an epidermal layer disposed on top of a non-cellular
layer disposed on top of a dermal layer, deposed on top of a
hypodermal layer.
[0110] The three-dimensional, engineered skin tissues, arrays, and
methods described herein, allow for the generation of multi
layered, compartmentalized engineered constructs. In certain
embodiments, a compartment is a discrete structure embedded within
the tissue or layer of the tissue that extends in the x, y and z
plane. In certain embodiments, a single compartment is embedded
within a single of layer. In certain embodiments, a single
compartment is embedded within a plurality of layers. In certain
embodiments, a plurality of compartments is embedded within a
plurality of layers. In certain embodiments, the skin tissues
comprise a plurality of compartments. In certain embodiments, the
compartments are in contact with each other. In certain
embodiments, the skin tissues comprise a plurality of compartments
disposed with separation of 10 .mu.m or more between the
compartments. In certain embodiments, the compartments comprise
epidermal, dermal, hypodermal or non-cellular bio-inks. In certain
embodiments, the compartment or plurality of compartments comprise
a specialized cell type such as sebaceous cells, follicular cells,
endothelial cells, muscle cells, smooth muscle cells, lymph nodes.
In certain embodiments, the compartment is embedded in the
epidermal layer. In certain embodiments, the compartment is
embedded in the dermal layer. In certain embodiments, the
compartment is embedded in the hypodermal layer. A compartment may
be spherical, cuboidal, rectangular, rhomboidal or any irregular
shape. In certain embodiments, the compartment extends through one
or more layers forming a tube that is open at the surface.
[0111] In certain embodiments, a compartment is a conglomeration of
a plurality of cells. In certain embodiments, a compartment is a
conglomeration of greater than 10 cells. In certain embodiments, a
compartment is a conglomeration of greater than 100 cells. In
certain embodiments, a compartment is a conglomeration of greater
than 500 cells. In certain embodiments, a compartment is a
conglomeration of greater than 1,000 cells. In certain embodiments,
a compartment is a conglomeration of greater than 5,000 cells. In
certain embodiments, a compartment is a conglomeration of greater
than 10,000 cells. In certain embodiments, a compartment is a
conglomeration of greater than 50,000 cells.
[0112] In certain embodiments, a compartment is greater than 1 cell
thick in its smallest dimension. In certain embodiments, a
compartment is greater than 10 cells thick in its smallest
dimension. In certain embodiments, a compartment is greater than 20
cells thick in its smallest dimension. In certain embodiments, a
compartment is greater than 50 cells thick in its smallest
dimension. In certain embodiments, a compartment is greater than
100 cells thick in its smallest dimension. In certain embodiments,
a compartment is greater than 500 cells thick in its smallest
dimension. In certain embodiments, a compartment is greater than
5000 cells thick in its smallest dimension.
[0113] In certain embodiments, a compartment is greater than 5
.mu.m thick in its smallest dimension. In certain embodiments, a
compartment is greater than 10 .mu.m thick in its smallest
dimension. In certain embodiments, a compartment is greater than 20
.mu.m thick in its smallest dimension. In certain embodiments, a
compartment is greater than 50 .mu.m thick in its smallest
dimension. In certain embodiments, a compartment is greater than
100 .mu.m thick in its smallest dimension. In certain embodiments,
a compartment is greater than 500 cells thick in its smallest
dimension.
[0114] In certain embodiments, the three-dimensional, engineered
skin tissues, arrays, and methods described herein comprise a basal
layer in contact with the dermal layer and the epidermal layer. In
certain embodiments, the basal layer is between the epidermal and
dermal layers. In certain embodiments, the basal layer comprises
basal keratinocytes. In certain embodiments, the basal layer is a
separate architecturally distinct layer. In certain embodiments,
the basal keratinocytes display increased expression of KRT14
(CK14) compared to non-basal keratinocytes. In certain embodiments,
the basal keratinocytes display increased expression of KRT5 (CK5)
compared to non-basal keratinocytes. In certain embodiments, the
basal layer is 1 cell thick. In certain embodiments, the basal
layer is greater than 2 cells thick. In certain embodiments, the
basal layer is greater than 3 cells thick. In certain embodiments,
the basal layer is greater than 5 cells thick. In certain
embodiments, the basal layer is greater than 10 cells thick. In
certain embodiments, the basal layer is greater than 50 cells
thick. In certain embodiments, the basal layer is less than 100
cells thick. In certain embodiments, the basal layer is less than
50 cells thick. In certain embodiments, the basal layer is less
than 10 cells thick.
[0115] In certain embodiments, the epidermal layer is a monolayer.
In certain embodiments, the epidermal layer is greater than 1 cell
thick. In certain embodiments, the epidermal layer is greater than
2 cells thick. In certain embodiments, the epidermal layer is
greater than 3 cells thick. In certain embodiments, the epidermal
layer is greater than 10 cells thick. In certain embodiments, the
epidermal layer is greater than 50 cells thick. In certain
embodiments, the epidermal layer is greater than 100 cells
thick.
[0116] In certain embodiments, the dermal layer is a monolayer. In
certain embodiments, the dermal layer is greater than 1 cell thick.
In certain embodiments, the dermal layer is greater than 2 cells
thick. In certain embodiments, the dermal layer is greater than 3
cells thick. In certain embodiments, the dermal layer is greater
than 10 cells thick. In certain embodiments, the dermal layer is
greater than 50 cells thick. In certain embodiments, the dermal
layer is greater than 100 cells thick.
[0117] In certain embodiments, the hypodermal layer is a monolayer.
In certain embodiments, the hypodermal layer is greater than 1 cell
thick. In certain embodiments, the hypodermal layer is greater than
2 cells thick. In certain embodiments, the hypodermal layer is
greater than 3 cells thick. In certain embodiments, the hypodermal
layer is greater than 10 cells thick. In certain embodiments, the
hypodermal layer is greater than 50 cells thick. In certain
embodiments, the hypodermal layer is greater than 100 cells
thick.
[0118] In some embodiments, the three-dimensional, engineered skin
tissues, arrays, and methods described herein, allow for unique
tissue architectures. Bio-inks are deposited to form layers or
discrete compartments. In certain embodiments, the epidermal tissue
and dermal tissue layers are separate, architecturally distinct
layers that are in direct contact or separated by 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20 .mu.m or more, including increments therein. In
certain embodiments, the separation is due to the secretion and
deposition of extracellular matrix between the two layers, which
for the purposes of this disclosure is considered contact. In
certain embodiments, a layer of non-cellular bio-ink is situated
between the epidermal and dermal layers. In certain embodiments,
the layer of non-cellular bio-ink is 10, 20, 30, 40, 50, 60, 70,
80, 90, or 100 .mu.m or more thick. In certain embodiments, the
epidermal cell layer is in contact with the dermal layer.
[0119] In certain embodiments, the epidermal cell layer or
compartment is in continuous contact with the dermal layer or
compartment. In certain embodiments, greater than 99% of the
epidermal cell layer or compartment is in contact with the dermal
layer or compartment. In certain embodiments, greater than 98% of
the epidermal cell layer or compartment is in contact with the
dermal layer or compartment. In certain embodiments, greater than
95% of the epidermal cell layer or compartment is in contact with
the dermal layer or compartment. In certain embodiments, greater
than 90% of the epidermal cell layer or compartment is in contact
with the dermal layer or compartment. In certain embodiments,
greater than 80% of the epidermal cell layer or compartment is in
contact with the dermal layer or compartment. In certain
embodiments, greater than 70% of the epidermal cell layer or
compartment is in contact with the dermal layer or compartment. In
certain embodiments, greater than 60% of the epidermal cell layer
or compartment is in contact with the dermal layer or compartment.
In certain embodiments, the epidermal cell layer is a monolayer. In
certain embodiments, the epidermal cell layer is greater than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 or more layers thick. In certain
embodiments, the epidermal cell layer is greater than 10, 20, 30,
40, 50, 60, 70, 80, 90, 100 or more layers thick. In certain
embodiments, the epidermal cell layer is a monolayer. In certain
embodiments, the dermal cell layer is greater than 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 or more layers thick. In certain embodiments, the
dermal cell layer is greater than 10, 20, 30, 40, 50, 60, 70, 80,
90, 100 or more cells thick. In certain embodiments, the epidermal
cell layer is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
layers thick. In certain embodiments, the epidermal cell layer is
less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more layers
thick. In certain embodiments, the dermal cell layer is less than
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more layers thick. In certain
embodiments, the dermal cell layer is less than 10, 20, 30, 40, 50,
60, 70, 80, 90, 100 or more cells thick.
[0120] In certain embodiments, the dermal cell layer or compartment
is in contact with the hypodermal cell layer or compartment. In
certain embodiments, the dermal cell layer or compartment is in
continuous contact with the hypodermal cell layer or compartment.
In certain embodiments, greater than 99% of the dermal cell layer
or compartment is in contact with the hypodermal layer or
compartment. In certain embodiments, greater than 98% of the dermal
cell layer or compartment is in contact with the hypodermal layer
or compartment. In certain embodiments, greater than 95% of the
dermal cell layer or compartment is in contact with the hypodermal
layer or compartment. In certain embodiments, greater than 90% of
the dermal cell layer or compartment is in contact with the
hypodermal layer or compartment. In certain embodiments, greater
than 80% of the dermal cell layer or compartment is in contact with
the hypodermal layer or compartment. In certain embodiments,
greater than 70% of the dermal cell layer or compartment is in
contact with the hypodermal layer or compartment. In certain
embodiments, greater than 60% of the dermal cell layer or
compartment is in contact with the hypodermal layer or compartment.
In certain embodiments, the hypodermal cell layer is a monolayer.
In certain embodiments, the hypodermal cell layer is greater than
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more layers thick. In certain
embodiments, the hypodermal cell layer is greater than 10, 20, 30,
40, 50, 60, 70, 80, 90, 100 or more layers thick. In certain
embodiments, the hypodermal cell layer is a monolayer. In certain
embodiments, the hypodermal cell layer is greater than 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 or more layers thick. In certain embodiments, the
dermal cell layer is greater than 10, 20, 30, 40, 50, 60, 70, 80,
90, 100 or more cells thick. In certain embodiments, the hypodermal
cell layer is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
layers thick. In certain embodiments, the hypodermal cell layer is
less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more layers
thick.
[0121] In certain embodiments, the thickness of the epidermal layer
of the three-dimensional, engineered skin tissues can be varied. In
certain embodiments, the thickness of the epidermal layer is
between about 20 and about 500 .mu.m. In certain embodiments, the
thickness of the epidermal layer is between about 20 and about 400
.mu.m. In certain embodiments, the thickness of the epidermal layer
is between about 20 and about 300 .mu.m. In certain embodiments,
the thickness of the epidermal layer is between about 20 and about
200 .mu.m. In certain embodiments, the thickness of the epidermal
layer is between about 50 and about 500 .mu.m. In certain
embodiments, the thickness of the epidermal layer is between about
100 and about 500 .mu.m. In certain embodiments, the thickness of
the epidermal layer is between about 100 and about 200 .mu.m. In
certain embodiments, the thickness of the epidermal layer is
greater than about 20 .mu.m. In certain embodiments, the thickness
of the epidermal layer is greater than about 30 .mu.m. In certain
embodiments, the thickness of the epidermal layer is greater than
about 40 .mu.m. In certain embodiments, the thickness of the
epidermal layer is greater than about 50 .mu.m. In certain
embodiments, the thickness of the epidermal layer is greater than
about 75 .mu.m. In certain embodiments, the thickness of the
epidermal layer is greater than about 100 .mu.m. In certain
embodiments, the thickness of the epidermal layer is greater than
about 125 .mu.m. In certain embodiments, the thickness of the
epidermal layer is less than about 500 .mu.m. In certain
embodiments, the thickness of the epidermal layer is less than
about 400 .mu.m. In certain embodiments, the thickness of the
epidermal layer is less than about 300 .mu.m. In certain
embodiments, the thickness of the epidermal layer is less than
about 200 .mu.m. In certain embodiments, the thickness of the
epidermal layer is about 150 .mu.m.
[0122] In certain embodiments, the thickness of the dermal layer of
the three-dimensional, engineered skin tissues can be varied. In
certain embodiments, the thickness of the dermal layer is between
about 10 and about 1000 .mu.m. In certain embodiments, the
thickness of the dermal layer is between about 100 and about 1000
.mu.m. In certain embodiments, the thickness of the dermal layer is
between about 200 and about 1000 .mu.m. In certain embodiments, the
thickness of the dermal layer is between about 300 and about 1000
.mu.m. In certain embodiments, the thickness of the dermal layer is
between about 400 and about 1000 .mu.m. In certain embodiments, the
thickness of the dermal layer is between about 100 and about 900
.mu.m. In certain embodiments, the thickness of the dermal layer is
between about 100 and about 800 .mu.m. In certain embodiments, the
thickness of the dermal layer is between about 100 and about 700
.mu.m. In certain embodiments, the thickness of the dermal layer is
between about 100 and about 600 .mu.m. In certain embodiments, the
thickness of the dermal layer is at least about 100 .mu.m. In
certain embodiments, the thickness of the dermal layer is at least
about 200 .mu.m. In certain embodiments, the thickness of the
dermal layer is at least about 300 .mu.m. In certain embodiments,
the thickness of the dermal layer is at least about 400 .mu.m. In
certain embodiments, the thickness of the dermal layer is less than
about 2000 .mu.m. In certain embodiments, the thickness of the
dermal layer is less than about 1500 .mu.m. In certain embodiments,
the thickness of the dermal layer is less than about 1000 .mu.m. In
certain embodiments, the thickness of the dermal layer is less than
about 900 .mu.m. In certain embodiments, the thickness of the
dermal layer is less than about 800 .mu.m. In certain embodiments,
the thickness of the dermal layer is less than about 700 .mu.m. In
certain embodiments, the thickness of the dermal layer is less than
about 600 .mu.m. In certain embodiments, the thickness of the
dermal layer is about 500 .mu.m.
[0123] In certain embodiments, the thickness of the hypodermal
layer of the three-dimensional, engineered skin tissues can be
varied. In certain embodiments, the thickness of the hypodermal
layer is greater than about 20 .mu.m. In certain embodiments, the
thickness of the hypodermal layer is greater than about 30 .mu.m.
In certain embodiments, the thickness of the hypodermal layer is
greater than about 40 .mu.m. In certain embodiments, the thickness
of the hypodermal layer is greater than about 50 .mu.m. In certain
embodiments, the thickness of the hypodermal layer is greater than
about 75 .mu.m. In certain embodiments, the thickness of the
hypodermal layer is greater than about 100 .mu.m. In certain
embodiments, the thickness of the hypodermal layer is greater than
about 200 .mu.m. In certain embodiments, the thickness of the
hypodermal layer is greater than about 300 .mu.m. In certain
embodiments, the thickness of the hypodermal layer is greater than
about 400 .mu.m. In certain embodiments, the thickness of the
hypodermal layer is greater than about 500 .mu.m. In certain
embodiments, the thickness of the hypodermal layer is greater than
about 125 .mu.m. In certain embodiments, the thickness of the
hypodermal layer is less than about 500 .mu.m. In certain
embodiments, the thickness of the hypodermal layer is less than
about 400 .mu.m. In certain embodiments, the thickness of the
hypodermal layer is less than about 300 .mu.m. In certain
embodiments, the thickness of the hypodermal layer is less than
about 200 .mu.m. In certain embodiments, the thickness of the
hypodermal layer is about 150 .mu.m.
[0124] In certain embodiments, the dermal layer is attached to a
biocompatible surface, and the epidermal layer is disposed on top
of the dermal layer. In certain embodiments, the epidermal layer
completely covers the dermal layer. In certain embodiments, the
epidermal layer covers greater than 95% of the dermal layer. In
certain embodiments, the epidermal layer or compartment covers
greater than 90% of the dermal layer or compartment. In certain
embodiments, the epidermal layer or compartment covers greater than
90% of the dermal layer or compartment. In certain embodiments, the
epidermal layer or compartment covers greater than 70% of the
dermal layer or compartment. In certain embodiments, the epidermal
layer or compartment covers greater than 60% of the dermal layer or
compartment. In certain embodiments, the epidermal layer or
compartment covers greater than 50% of the dermal layer or
compartment. In certain embodiments, the epidermal layer or
compartment covers greater than 40% of the dermal layer or
compartment. In certain embodiments, the epidermal layer or
compartment covers greater than 30% of the dermal layer or
compartment. In certain embodiments, the epidermal layer or
compartment covers greater than 20% of the dermal layer or
compartment. In certain embodiments, the epidermal layer or
compartment covers greater than 10% of the dermal layer or
compartment.
[0125] In certain embodiments, the hypodermal layer is attached to
a biocompatible surface, and the dermal layer is disposed on top of
the dermal layer. In certain embodiments, the dermal layer
completely covers the hypodermal layer. In certain embodiments, the
dermal layer covers greater than 95% of the hypodermal layer. In
certain embodiments, the dermal layer covers greater than 90% of
the hypodermal layer. In certain embodiments, the dermal layer
covers greater than 90% of the dermal layer. In certain
embodiments, the dermal layer covers greater than 70% of the
hypodermal layer. In certain embodiments, the dermal layer covers
greater than 60% of the hypodermal layer. In certain embodiments,
the dermal layer covers greater than 50% of the hypodermal
layer.
[0126] In some aspects the three-dimensional, engineered skin
tissues are substantially flat. In some aspects the skin tissues
are flat with less than 10% curvature. In some aspects the skin
tissues are substantially flat with less than 20% curvature. In
some embodiments, the surface area of the skin tissues is at least
0.01 cm.sup.2. In some embodiments, the surface area of the skin
tissues is at least 0.02 cm.sup.2. In some embodiments, the surface
area of the skin tissues is at least 0.03 cm.sup.2. In some
embodiments, the surface area of the skin tissues is at least 0.04
cm.sup.2. In some embodiments, the surface area of the skin tissues
is at least 0.05 cm.sup.2. In some embodiments, the surface area of
the skin tissues is at least 0.06 cm.sup.2. In some embodiments,
the surface area of skin tissues is at least 0.07 cm.sup.2. In some
embodiments, the surface area of the skin tissues is at least 0.08
cm.sup.2. In some embodiments, the surface area of the skin tissues
is at least 0.09 cm.sup.2. In some embodiments, the surface area of
the skin tissues is at least 0.10 cm.sup.2. In some embodiments,
the surface area of the renal tubule model is at least 0.11
cm.sup.2. In some embodiments, the surface area of the skin tissues
is at least 0.12 cm.sup.2. In some embodiments, the surface area of
the skin tissues is less than 0.2 cm.sup.2. In some embodiments,
the surface area of the skin tissues is less than 0.4 cm.sup.2. In
some embodiments, the surface area of the skin tissues is less than
0.5 cm.sup.2. In some embodiments, the surface area of the skin
tissues is less than 0.8 cm.sup.2. In some embodiments, the surface
area of skin tissues is less than 1.0 cm.sup.2. In some
embodiments, the surface area of skin tissues is less than 2.0
cm.sup.2. In some embodiments, the surface area of skin tissues is
less than 3.0 cm.sup.2. In some embodiments, the surface area of
skin tissues is less than 4.0 cm.sup.2. In some embodiments, the
surface area of skin tissues is less than 5.0 cm.sup.2.
[0127] In certain embodiments, the tissue can be any suitable shape
such as square, oval, ellipsoid, circular, trapezoidal, rhomboidal,
spherical, cuboidal, and the like.
Methods of Manufacture
[0128] Referring to FIG. 3, for example, in a particular
embodiment, continuous deposition bioprinting techniques are used
to print a layered tissue onto a collagen-coated printing surface.
In this embodiment, dermal cells are bioprinted onto a transwell
surface to form a dermal layer. Subsequently, epidermal cells are
bioprinted on top of the dermal cells to form an epidermal layer.
Finally, the layered tissue is allowed to mature in a cell culture
environment.
[0129] Referring to FIG. 4, in a particular embodiment, an
engineered skin tissue containing dermal and epidermal layers
bioprinted using continuous deposition techniques was allowed to
mature for 48 hours post-printing.
[0130] An advantage of the engineered tissues and methodologies
described herein is that they allow retention of the shape of the
structure without compromising the functionality of the original
cell types. The shape of the bioprinted structure is advantageously
maintained by multiple approaches. In Example 1, the printed
bio-ink structure utilizes Novogel.RTM. 3.0, which is cross-linked
at the time of the printing to maintain shape and lyase treated at
a later time point while maturing at 37.degree. C. Because the
cross-linking step involves exposure to high concentrations of
calcium ions, which could impact keratinocyte biology, we sought to
separate this cross-linking step from the deposition of the
epidermal layer. The invention also incorporates a novel aerosol
spray printing method into a 3D tissue model. The aerosol spray
approach provides a unique method compared to the continuous
deposition method in that it allows the creation of a thinner
layer, and allows you to readily deposit material onto an existing
tissue layer after a period of maturation. This is advantageous
because it may produce a tissue that better mimics native tissue in
vivo. This can also be advantageous because it can reduce the
number of cells required and allow for bioprinting with limited
cell populations. This aerosol spray method can be applied to
create multiple layers at multiple time points. For example, this
method could be used for spraying first with undifferentiated
keratinocytes followed by spraying with differentiated
keratinocytes to better mimic native skin (FIG. 5).
[0131] The aerosol spray bioprinting techniques described herein
allow for the spray of materials that include, for example, a cell
suspension, media, bio-ink, biosupport material, or a combination
thereof. In Example 1, the aerosol spray approach is utilized to
spray a thin layer of epidermal cells. In some embodiments, the
engineered tissues and methodologies described herein highlight the
ability to spray single cells at a resolution of one cell layer
thickness and the ability to spray cell aggregates. The sprayed
layer could, however, also be modified by changing parameters
including but not limited to spray material velocity, distance,
time, volume, and viscosity. For the creation of the epidermal
layer, cells are optionally sprayed onto other bioprinted layers to
result in a full-thickness model, or directly onto transwell or
other matrix coated surfaces to specifically generate an epidermal
model. The spray method is optionally utilized to embed sprayed
material into a soft surface such as biosupport material or
Novogel.RTM.. For example, a dermal layer could be created by
spraying fibroblasts into a collagen gel (FIG. 6). In some
embodiments, this approach generates a dermal layer that more
closely resembles native dermis, where a more sparse cellular
density is observed than is usually achieved by continuous
deposition methods.
[0132] The aerosol spray method is unique when compared to
continuous deposition printing in that it does not require a flat
printing surface, such as a transwell membrane, to zero the initial
printing position in the x, y, and z-axes. The aerosol spray method
is optionally used to apply a layer to an uneven surface such as a
structure previously printed by continuous deposition. For example,
in Example 2 the aerosol spray bioprinting methodologies described
herein are utilized to spray a thin layer of epidermal cells onto
previously printed layer of dermal cells. In some embodiments, this
temporal spacing allows the initial layer to express certain
proteins that enable adherence and proper stratification of the
subsequent epidermal layer.
[0133] Regardless of the printing method used, a variety of factors
are optionally modified to promote proliferation and/or
differentiation of printed tissue cells. In some cases, dermal
media, epidermal media, or a combination of dermal and epidermal
media is added to the skin tissue constructs. In addition, the
media composition is optionally changed at different points in the
tissue lifetime to promote the desired biology. The tissue
constructs are optionally moved to an air liquid interface or
subjected to atmospheric changes such as modification of humidity
or CO.sub.2. A hypothetical experimental design combining both
printing approaches is shown in FIG. 7.
[0134] In certain embodiments, the three-dimensional, engineered,
biological skin tissues disclosed herein are produced by an
additive manufacturing process. The additive manufacturing process
for three-dimensional, engineered, biological skin tissues herein
allows customized fabrication of three-dimensional, engineered,
biological skin tissues for in vitro and therapeutic purposes. This
is significant in that the tissues are fabricated due to a user
specified design. In certain embodiments, the three-dimensional,
engineered, biological skin tissues contain only the cells that the
user specifies (e.g., uses as inputs to the additive manufacturing
process). In certain embodiments, three-dimensional, engineered,
biological skin tissues contain only the cell types that the user
specifies. In certain embodiments, the three-dimensional,
engineered, biological skin tissues contain only the number of
cells or concentration of cells that the user specifies. In certain
embodiments, the three-dimensional, engineered, biological skin
tissues contain cells that have been treated with a small molecule,
therapeutic molecule, or therapeutic substance before or during
fabrication. In certain embodiments, the three-dimensional,
engineered, biological skin tissues contain biocompatible or tissue
culture plastics, biocompatible synthetic polymers, cross linkable
gels, reversibly cross-linked gels and other non-cellular
constituents.
Bioprinting
[0135] In some embodiments, at least one component of the
engineered skin tissues/constructs, 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 support 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 adhesion of cells
or multicellular bodies which, in some cases, is similar to
self-assembly phenomena in early morphogenesis.
[0136] 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.
[0137] 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.
[0138] In some embodiments, the method of bioprinting is
discontinuous. A non-limiting example of discontinuous bioprinting
is when bio-ink or cells are dispensed, and then the flow of
bio-ink or cells is stopped, paused for a certain amount of time,
and then started again. This can allow for different bio-inks or
cells, or the same bio-inks or cells to be layered with a delay in
printing of the layers. In some embodiments, the discontinuous
bioprinting is achieved using an aerosol spray type of bioprinting,
wherein cells are applied to an existing tissue layer or surface
using an aerosol spray technology. In some embodiments, a single
layer or plurality of layers of dermal cells or bio-inks are
deposited, followed by a temporal delay in deposition of a single
layer or plurality of layers epidermal cells or bio-inks. In some
embodiments, the deposition of the epidermal cells is by an aerosol
spray.
[0139] Any of the different bio-inks of this disclosure can be
deposited by various techniques to form layers of the
three-dimensional, engineered, biological skin tissue. Any of the
layers can be deposited by extrusion (continues or discontinuous),
spraying (ink jettingor aerosol spraying). In certain embodiments,
the hypodermal bio-ink is deposited by extrusion onto a surface. In
certain embodiments, the hypodermal bio-ink is deposited by
extrusion onto a surface. In certain embodiments, the dermal
bio-ink is deposited by extrusion onto a surface. In certain
embodiments, the epidermal bio-ink is deposited by extrusion onto a
surface. In certain embodiments, a non-cellular matrix bio-ink is
deposited by extrusion onto a surface. In certain embodiments, the
hypodermal bio-ink is deposited by spraying onto a surface. In
certain embodiments, the hypodermal bio-ink is deposited by
spraying onto a surface. In certain embodiments, the dermal bio-ink
is deposited by spraying onto a surface. In certain embodiments,
the epidermal bio-ink is deposited by spraying onto a surface. In
certain embodiments, the non-cellular matrix bio-ink is deposited
by spraying onto a surface. In certain embodiments, the hypodermal
bio-ink is not deposited by extrusion onto a surface. In certain
embodiments, the hypodermal bio-ink is not deposited by extrusion
onto a surface. In certain embodiments, the dermal bio-ink is not
deposited by extrusion onto a surface. In certain embodiments, the
epidermal bio-ink is not deposited by extrusion onto a surface. In
certain embodiments, a non-cellular matrix bio-ink is not deposited
by extrusion onto a surface. In certain embodiments, the hypodermal
bio-ink is not deposited by spraying onto a surface. In certain
embodiments, the hypodermal bio-ink is not deposited by spraying
onto a surface. In certain embodiments, the dermal bio-ink is not
deposited by spraying onto a surface. In certain embodiments, the
epidermal bio-ink is not deposited by spraying onto a surface. In
certain embodiments, the non-cellular matrix bio-ink is not
deposited by spraying onto a surface.
[0140] In certain embodiments, deposition of the epidermal bio-ink
occurs after deposition of the dermal bio-ink. In certain
embodiments, deposition of the epidermal bio-ink occurs before
maturation of the deposited dermal bio-ink. In certain embodiments,
deposition of the epidermal layer is temporally delayed before it
is deposited on the dermal bio-ink. In certain embodiments, the
delay is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, milliseconds. In
certain embodiments, the delay is greater than 10 milliseconds. In
certain embodiments, the delay is greater than 20, 30, 40, 50, 60,
70, 80, 90 or 100, milliseconds. In certain embodiments, the delay
is greater than 200, 300, 400, 500, 600, 700, 800, 900 or 1000,
milliseconds. In certain embodiments, the delay is greater than 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 seconds. In certain embodiments, the
delay is greater than 10, 20, 30, 40, 50, or 60 seconds. In certain
embodiments, the delay is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 minutes. In certain embodiments, the delay is greater than
10, 20, 30, 40, 50, or 60 minutes. In certain embodiments, the
delay is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In certain
embodiments, the delay is greater than 1, 2, 3, 4, 5, 6, or 7 days.
In certain embodiments, the delay is greater than 1, 2, 3, or 4
weeks. In certain embodiments, the delay is less than 1, 2, 3, 4,
5, 6, 7, 8, 9, milliseconds. In certain embodiments, the delay is
less than 10 milliseconds. In certain embodiments, the delay is
less than 20, 30, 40, 50, 60, 70, 80, 90 or 100, milliseconds. In
certain embodiments, the delay is less than 200, 300, 400, 500,
600, 700, 800, 900 or 1000, milliseconds. In certain embodiments,
the delay is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 seconds. In
certain embodiments, the delay is less than 10, 20, 30, 40, 50, or
60 seconds. In certain embodiments, the delay is less than 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 minutes. In certain embodiments, the delay
is less than 10, 20, 30, 40, 50, or 60 minutes. In certain
embodiments, the delay is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In
certain embodiments, the delay is less than 1, 2, 3, 4, 5, 6, or 7
days. In certain embodiments, the delay is less than 1, 2, 3, or 4
weeks.
[0141] In certain embodiments, the skin tissue also comprises a
test substance or agent, and can be administered to the tissue in
many ways as shown in FIG. 26, the test substance can be applied to
the apical, ventral, basal, or lateral surface of any single layer
or any plurality of layers bioprinted. The test substance can be
applied to the entirety of the surface or a portion of the surface.
The test substance can be added between two layers that are the
same cell type or a different cell type, embedded within a single
layer or plurality of layers, or mixed homogenously or
heterogeneously throughout a single layer or a plurality of layers.
The test substance can be applied via an aerosol spray type
mechanism, an extrusion mechanism, a syringe a pipette tip, or a
blunted object.
[0142] This invention discloses the automated administration of
substances for toxicology testing through utilization of a
bioprinting platform including but not limited to ink jet
aerosolized spray, dispense nozzle, and continuous deposition.
Bioprinting allows for spatially defined, precise deposition of
predetermined volumes and geometries onto printing surfaces. In one
embodiment, tissues are printed onto surfaces containing printed
test substances. In a second embodiment, substances are
administered by printing onto printed tissues. In some instances
test substances can be applied topically to a tissue simultaneously
during printing, in other instances immediately following printing,
and in yet other instances at a later time point to mature tissue.
For example, a test substance can be added to a mature tissue to
model transdermal administration in which the substance must pass
through fully formed stratum corneum with barrier function. In a
third embodiment, substances can be incorporated into a tissue. In
some cases, a test substance can be added as a layer between
printed tissues. In other cases, substances are printed as a
homogenous mixture within any layer of the skin tissue, or within
all layers of the skin tissue. In yet other cases, a substance can
be embedded within a tissue layer. For example, a test substance
can be embedded into the dermal layer of a full thickness skin
tissue by syringe deposition to model a parenteral subcutaneous
injection.
[0143] Test substances may be applied directly to the apical
surface of the tissue to model transdermal delivery and test
barrier function, or administered to ventral or lateral sides of
the tissue to model permeation or distribution as a gradient. In
other applications, tissues are completely immersed in the
substance. In some instances, a bioprinted matrix or membrane may
act as an adhesive or aid to administer, dispense or disperse a
test substance similar to a transdermal patch. In other instances
addition or adherence can be aided in combination or separately by
a patch of a non-bioprinted material such as gauze or nylon mesh.
Substances can be also administered manually, for example, as
dispensed by pipet tip, swabbed, or applied by a blunted
object.
[0144] Test substances can be liquid, including solutions,
suspensions, and emulsions. Substances can also be solids, such as
powders and granules, or semi-solids such as pastes. Substances
tested can be hydrous or anhydrous with varying levels of viscosity
and administered in the form of but not limited to a liquid, oil,
gel, foam, ointment, or cream. Substances may be applied
aerosolized into a liquid aerosol such as a spray or fog.
Substances may also be administered as a solid aerosol such as a
smoke or dust. Substances may also be administered combined with a
Novogel.RTM.. Any of these test substances may be applied in an
automated way, by bioprinting or manually.
[0145] In some embodiments, the test substance is any substance
requiring toxicology, pharmaceutical or cosmetic testing. The test
substance is a composition containing any whole, part, active or
inactive ingredient of a chemical mixture, irritant, chemical,
pharmaceutical, alcohol, lipid, phospholipid, acid, base, peroxide,
oxidizing agent, reducing agent, detergent, surfactant,
nutraceutical, vitamin, pro-vitamin, mineral, amino acid, DNA, RNA,
protein, enzyme, allergen, pet allergen, plant based allergen,
mold, dust, insect venom, virus, bacteria, fungus, immunological
adjuvant, antibiotic, antifungal, sunscreen, insect repellant,
cosmetic, botanical, chap stick, lipstick, mascara, eye shadow,
foundation, powder, make up remover, soap, body wash, face wash,
hand soap, dishwashing soap, shampoo, cologne, perfume, aftershave,
shaving lotion, shaving gel, shaving cream, lubricant, conditioner,
hair-dye, hair remover, moisturizer, anti-wrinkle cream, laundry
detergent, fabric softener or latex. In certain embodiments, the
test substance is not chemical in nature, examples include, but are
not limited to, light, sunlight, ultraviolet light, X-rays,
electromagnetic radiation, electrical impulses applied to or in the
vicinity of the tissue, lasers, heat, cold, acoustic waves, or
mechanical stress. In certain embodiments, the test substance is a
plurality of substances applied simultaneously or in sequence. The
composition applied can be any pharmaceutically, dermatologically
or cosmetically acceptable substance. The substance can be a
lotion, ointment, aqueous solution, aerosol, mist, suspension,
colloid, tincture, alcohol based or lipid based solution. In
certain embodiments, the composition contains DMSO.
Pre-Formed Scaffold
[0146] In some embodiments, disclosed herein are engineered,
engraftable skin tissues 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.
[0147] In some embodiments, the engineered skin tissues/constructs
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 skin 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 skin 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.
[0148] In some embodiments, the engineered skin 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.
Biocompatible Surfaces
[0149] In some embodiments, the engineered skin tissues/constructs
are secured to a biocompatible surface on one or more sides. In
some embodiments, the engineered skin tissues/constructs are
secured to a biocompatible surface 1, 2, 3, 4, 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 surface. In some
embodiments, the pore size of the surface can be greater than 0.2
.mu.m. In some embodiments, the pore size of the surface can be
greater than 1 .mu.m. In some embodiments, a tissue is suitably
secured to a biocompatible surface by cell-attachment to a porous
membrane. In some embodiments, the engineered skin
tissues/constructs 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. In certain embodiments, the
biocompatible surface is flexible. In certain embodiments, the
biocompatible surface is non-static (in motion) at the time of
bioprinting. In certain embodiments, the biocompatible surface is
not flat. The biocompatible surface could be a mold or form shaped
like a human or other mammalian body part, or is curved.
[0150] 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 skin tissues/constructs are subjected
to shear force, caused by fluid flow. In various embodiments, the
engineered skin tissues are subjected to shear force on 1, 2, 3, 4,
or more sides. In further embodiments, the engineered skin
tissues/constructs are subjected to recirculation, perfusion, or
agitation of the liquid nutrients that contact the tissues on one
or more exposed surfaces.
Arrays
[0151] In some embodiments, disclosed herein are arrays of
engineered skin tissues/constructs. 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.
[0152] In some embodiments, the engineered skin tissues/constructs
each exist in a well of a biocompatible multi-well container. 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.RTM.. 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.
[0153] In some embodiments, the arrays of engineered tissues,
including skin tissues/constructs, 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, 500 or 1,000 elements, including increments
therein. In further embodiments, each element comprises one or more
cells, multicellular aggregates, tissues, organs, or combinations
thereof.
[0154] In some embodiments, the arrays of engineered tissues,
including skin tissues/constructs, 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 medium- or
high-throughput biological assay or screening methods or
devices.
[0155] 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 skin tissues are
included in an array to facilitate investigation of a particular
disease or condition. In some embodiments, one or more specific
engineered skin 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 skin
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, and 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.
[0156] 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.
[0157] In certain embodiments, tissues within the array are spaced
at regular intervals in a repeating pattern. In certain
embodiments, tissues within the array are spaced at least 10 .mu.m
but no more than 1000 .mu.m apart. In certain embodiments, tissues
within the array are spaced at least 10 .mu.m but no more than 500
.mu.m apart. In certain embodiments, tissues within the array are
spaced at least 10 .mu.m but no more than 200 .mu.m apart. In
certain embodiments, tissues within the array are spaced at least
20 .mu.m but no more than 1000 .mu.m apart. In certain embodiments,
tissues within the array are spaced at least 50 .mu.m but no more
than 1000 .mu.m apart. In certain embodiments, tissues within the
array are spaced at least 100 .mu.m but no more than 1000 .mu.m
apart. In certain embodiments, tissues within the array are spaced
at least 20 .mu.m apart. In certain embodiments, tissues within the
array are spaced at least 50 .mu.m apart. In certain embodiments,
tissues within the array are spaced at least 100 .mu.m apart.
In Vitro Assays
[0158] In some embodiments, the engineered skin tissues 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.
[0159] In various embodiments, the engineered skin tissues 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 lipids, proteins or mRNAs. In various further
embodiments, the engineered skin tissue and arrays are for use in
assays to detect or measure one or more of: barrier function,
molecular binding (including radio ligand binding), molecular
uptake, activity (e.g., enzymatic activity and receptor activity,
etc.), gene expression, protein expression, protein modifications
(non-limiting examples include: phosphorylation, ubiquitination,
acetylation, glycosylation, lipidation, etc.) receptor agonism,
receptor antagonism, cell signaling, apoptosis, DNA damage, stress
response, cohesion, permeability, inflammation, pigmentation,
chemosensitivity, transfection, cell migration, chemotaxis, cell
viability, cell proliferation, safety, efficacy, metabolism,
toxicity, infectivity, and abuse liability. In various embodiments,
the skin tissue are for toxicology, pharmaceutical or cosmetic
testing.
[0160] In some embodiments, the engineered skin tissues and arrays
are for use in immunoassays. Immunoassays include, for example,
flow cytometry, high throughput or low throughput image analysis,
immunoprecipitation, radio-immunoassay (RIA), ELISA, western blot,
homogenous assays, such as AlphaLISA.TM. and related technologies
that rely on time resolved fluorescence or fluorescence resonance
energy transfer (FRET). 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.
[0161] In some embodiments, the engineered skin tissues and arrays
are for use in metabolic conversion or permeability assays. Assays
include, for example, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl
tetrazolium bromide (MTT), resazurin, lactate dehydrogenase (LDH),
calcein AM substrates, related dyes, and technologies that rely on
fluorescence or absorbance.
[0162] In some embodiments, the engineered skin tissue 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.
[0163] 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 engineered
skin tissue/construct 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.
[0164] 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.
[0165] In some embodiments, the engineered skin tissue and arrays
are for use in cell-based screening. In further embodiments, the
cell-based screening is for one or more infectious diseases such as
viral, fungal, bacterial or parasitic infection. In further
embodiments, the cell-based screening is for skin cancer, including
melanoma, basal cell carcinoma, and squamous cell carcinoma. In
further embodiments, the cell-based screening is for dermatitis,
including, atopic dermatitis, contact dermatitis, dermatitis
herpetiformis, neurodermatitis and seborrheic dermatitis. In
further embodiments, the cell-based screening is for psoriasis. In
further embodiments, the cell-based screening is for one eczema,
including xerotic eczema, discoid eczema, venous eczema, and
autoeczematization. In further embodiments, the cell-based
screening is for keratosis including actinic keratosis (also known
as solar keratosis), hydrocarbon keratosis, keratosis pilaris (KP,
also known as follicular keratosis), and seborrheic keratosis. In
further embodiments, the cell-based screening is for acne. In other
embodiments, the engineered skin tissues and arrays are for use in
the study of cancer initiation, progression, or metastasis. In
still further embodiments, the engineered skin tissues and arrays
are for use in the study of the interaction of other cell types,
such as cancer cells, pathogen-bearing cells, pathogenic cells,
immune cells, blood-derived cells, or stem/progenitor cells.
[0166] In some embodiments, the constructs or arrays thereof are
for use in assessing the performance of biologics, including
antibodies, mammalian cells, bacteria, biologically-active
proteins, hormones, etc. In some embodiments, the construct or
arrays thereof are for use to detect, quantify, and study
immunologic sampling by Langerhans cells, including the effects of
gram-negative or gram-positive antigen-stimulated signaling from
Langerhans cells, macrophages, T cell or B-cells to bordering skin
cells. In other embodiments, the skin constructs or arrays thereof
are useful in the study of cancer initiation, progression, or
metastasis. In other embodiments, the skin constructs or arrays
thereof are useful in the study of cell-cell and cell-tissue
interactions between the mammalian skin cells/tissue comprising the
construct and one or more additional cell types, including but not
limited to pathogen-bearing cells, living pathogenic cells, cancer
cells, immune cells, blood cells, stem/progenitor cells, or
genetically-manipulated cells.
[0167] In some embodiments, the array comprises engineered skin
tissue constructs and additional tissue constructs. In further
embodiments, the skin tissue construct is in direct contact with an
additional tissue construct on one or more surfaces. In still
further embodiments, the skin tissue is connected to one or more
additional tissues constructs or cells via a fluid path or common
fluid reservoir. In still further embodiments, the liquid media
that contacts the engineered skin tissue construct contains living
mammalian cells such as immune cells, blood-derived cells, or
tumor-derived cells. In other embodiments, the liquid media that
contacts the engineered skin tissue construct contains bacteria,
fungi, viruses, parasites, or other pathogens.
Therapeutic Applications
[0168] In certain embodiments, the skin tissue of the current
application is for use in treating a subject with a skin condition.
In certain embodiments, the three-dimensional skin tissue is for
engraftment to a subject. The skin condition could be any condition
for which skin grafts are utilized. The condition could be due to
trauma such as burns caused by heat or chemicals. The condition
could be caused by trauma that results in an open wound or the
reopening of a previously closed wound. The condition could be
caused by trauma that results in the removal of skin. The condition
could be a skin cancer. The condition could be due to infection
such as necrotizing fasciitis or purpura fulminans. The condition
could be caused by skin necrosis. The condition could be cosmetic.
The condition could be a cosmetic defect. The condition could be
due to aging. In certain embodiments, the three-dimensional skin
tissues are for use in procedures that aid wound healing.
[0169] In certain embodiments, the skin cells used in the
three-dimensional engineered skin tissue are derived from the
subject being treated (e.g., autologous). In certain embodiments,
the skin cells used in the three-dimensional engineered skin tissue
are from a donor considered to be histocompatible. In certain
embodiments, the skin cells used in the three-dimensional
engineered skin tissue are from a donor considered to be
non-histocompatible. In certain embodiments, the three-dimensional
engineered skin tissue is considered to be isogeneic, allogeneic or
xenogeneic to the recipient. In certain embodiments, the cells used
in the three-dimensional engineered skin tissue are pluripotent
cells including, stem cells, induced pluripotent stem cells,
embryonic stem cells, mesenchymal stem cells, or adult stem
cells.
[0170] In some embodiments, the cells utilized in the
three-dimensional engineered skin tissue are modified. In some
embodiments, the cells utilized in the three-dimensional engineered
skin tissue are modified to reduce rejection of the graft by the
immune system. In some embodiments, the cells utilized in the
three-dimensional engineered skin tissue are modified to promote
histocompatibility between the three-dimensional engineered skin
tissue and the recipient subject. In some embodiments, the cells
utilized in the three-dimensional engineered skin tissue are
modified to correct a congenital defect. In some embodiments, the
modification of the cells utilized in the three-dimensional
engineered skin tissue is biological, chemical or physical. In some
embodiments, the modification of the cells utilized in the
three-dimensional engineered skin tissue is genetic. In some
embodiments, the genetic modification is the result of expression
of a transgene, open reading frame, short hairpin RNA (shRNA),
small interfering RNA (siRNA) or micro RNA (miRNA).
[0171] In some embodiments, a therapeutic substance is used to
treat the cells before bioprinting. In some embodiments, the
therapeutic substance is bioprinted with the cells, and included in
the three dimensional engineered skin tissue. In some embodiments,
the three-dimensional skin tissue is treated with a therapeutic
substance sometime after bioprinting. In some embodiments, the
therapeutic substance is an antibiotic, an antiviral, an
antifungal, an anti-inflammatory, an immunosuppressant, an
analgesic, an opiate, a vasoconstrictor, a vasodilator, a steroid,
or a vitamin mixture. In certain embodiments, the therapeutic
substance can also be any substance used to protect skin or promote
its attachment or ability to thrive at a site of engraftment. These
include but are not limited to skin protectants, moisturizers,
adhesives, (biodegradable or non-biodegradable), physical barriers,
porous membranes, or non-porous membranes, gels or scaffolds.
[0172] In certain embodiments, the skin tissue also comprises a
therapeutic substance, and can be administered to the tissue in
many ways as shown in FIG. 27, the therapeutic substance can be
applied to the apical, ventral, basal, or lateral surface of any
single layer or any plurality of layers bioprinted. The therapeutic
substance can be applied to the entirety of the surface, or a
portion of the surface. The therapeutic substance can be added
between two layers that are the same cell type or a different cell
type, embedded within a single layer or plurality of layers, or
mixed homogenously or heterogeneously throughout a single layer or
a plurality of layers. The therapeutic substance can be applied via
an aerosol spray type mechanism or an extrusion mechanism. In other
applications, tissues are completely or partially immersed in the
therapeutic substance. In some embodiments, the tissue can be
applied by suturing, stapling or the use of adhesives or films. In
some instances, a bioprinted matrix or membrane may act as an
adhesive or aid to administer, dispense or disperse a therapeutic
substance similar to a transdermal patch. In other instances
addition or adherence can be aided in combination or separately by
a patch of a non-bioprinted material such as gauze or nylon mesh.
Substances can be also administered manually, for example, as
dispensed by pipet tip, swabbed, or applied by a blunted object.
Therapeutic substances can be liquid, including solutions,
suspensions, and emulsions. Therapeutic substances can also be
solids, such as powders and granules, or semi-solids such as
pastes. Therapeutic substances can be hydrous or anhydrous with
varying levels of viscosity and administered in the form of but not
limited to a liquid, oil, gel, foam, ointment, or cream. Substances
may be applied aerosolized into a liquid aerosol such as a spray or
fog. Substances may also be administered as a solid aerosol such as
a smoke or dust. Substances may also be administered combined with
a hydrogel such as Novogel.RTM..
[0173] The disclosure herein includes systems for in vitro
screening. The disclosure herein includes 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 skin tissues and/or organs for engraftment or use in
generation of cell-based tools for research and development, such
as in vitro assays. In further embodiments, the engineered skin
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 skin tissues and/or organs and arrays thereof are
produced and utilized to conduct biological assays and/or drug
screening as a service.
EXAMPLES
[0174] The following illustrative examples are representative of
embodiments of the software applications, systems, and methods
described herein and are not meant to be limiting in any way.
Example 1
Bioprinting Full Thickness Skin Tissue by Continuous Deposition
Using Dermal Bio-Ink Containing Alginate and Epidermal Bio-Ink
Deposition by Aerosol Spray Method
Procedures
[0175] Bio-ink was generated by a cellular mixture of 100% primary
adult human dermal fibroblasts (HDFa) in 6% gelatin and 1% alginate
(Novogel.RTM. 3.0) in a concentration of 150 million cells per
milliliter. Three-dimensional bio-ink constructs were printed by
continuous deposition using the Novogen Bioprinter.RTM. platform in
a 4 mm.times.4 mm.times.0.5 mm base sheet with a 1 mm wall
bordering the top to create a dermal structure resembling a cup.
One tissue construct was printed per transwell in a 6 well plate.
The transwell printing surface contained a polytetrafluoroethylene
(PTFE) membrane coated with equimolar mixture of types I and III
collagen (bovine) with pores 3 .mu.m in size. Following printing,
constructs were immediately cross-linked by submerging in 5 ml of
50 mM calcium chloride for 2-5 minutes. Calcium chloride was then
aspirated and constructs were submerged in 5 ml of fibroblast
growth media (DMEM containing 10% FBS and P/S/A). Constructs were
allowed to mature for 24 hours in a non-humidified 37.degree. C.
incubator. After 24 hours, dermal tissue constructs were removed
from the incubator and placed in a BSC hood. Media was aspirated
immediately before aerosol spray application. Epidermal bio-ink was
generated by a cell suspension mixture of 90% primary adult human
epidermal keratinocytes (HEKa) and 10% primary adult human
epidermal melanocytes (HEMa) in a concentration of 1 million cells
per milliliter media. Media used contained 90% keratinocyte growth
media and 10% melanocyte media. Cells were dispensed into a sterile
glass vial in a BSC hood in an aseptic manner and agitated manually
to maintain suspension. The vial containing the cell suspension was
attached to two tubes running through a tightly sealed lid. One set
of tubing connected the volume of cell suspension to a spray nozzle
inside the hood. Another set of tubing running from the inside of
the vial lid connected to a compressed air tank outside the hood
adjusted to 23-25 psi. The diameter of the spray was controlled by
the height of the spray nozzle. The height of the spray nozzle was
adjusted manually by attaching the tubing to an adjustable stand.
The height was set at approximately 3 cm to allow for the height of
a 6 well plate. The spray diameter was approximately 2.5 cm, or the
width of one well in the plate. The rate of flow of the spray
nozzle was controlled digitally by pulsed dispenses of 100 ms or
200 ms per spray. Well plates containing bioprinted dermal
fibroblast tissue constructs were removed from the 37.degree. C.
incubator after 24 hours. Construct media was aspirated. Each well
containing a dermal construct was individually placed directly
under the spray nozzle. Applications of single, double, or multiple
pulses at 100 ms or 200 ms were sprayed onto the surface of the
construct. As a control, spray was also administered to the
transwell membrane alone. After aerosol spray, 2 mls of media was
added to the outer area of the transwell basket. The media used for
subsequent growth and maintenance of the skin tissue was a 50:40:10
ratio of HDFa:HEKa:HEMa media. The volume added was sufficient to
collect at the base of the printed structure but not to submerge
the structure. The spray volume was measured post print by
collecting the dispensed volume into a 1.5 ml tube. Spray volume
averaged 23 .mu.l per spray pulse at 200 ms and 8.7 .mu.l per spray
at 100 ms. Viability was measured at 90% by trypan blue exclusion
assay. Cell numbers averaged 1.4 million cells per ml which were
back calculated to 32,200 cells per spray at 200 ms and 12,180
cells per spray at 100 ms. Tissues were treated with 0.34 mg/ml
alginate lyase for 4 hours on day 3 (48 hours post aerosol spray
application) in a volume of 3 ml per well. Media was added to the
outer area of the transwell. The meniscus of the volume of lyase
media was sufficient reach the top of the construct side without
submerging the construct. Following lyase treatment, media was
aspirated and 1 ml of fresh media was added to the outer area of
each well bringing the tissue constructs to an air liquid
interface. Media was changed daily at 1 ml per well subsequently
for up to 12 days. After incubation, the constructs were fixed in
2% paraformaldehyde (PFA) for histology.
Results
[0176] Bioprinted dermal constructs were grown in fibroblast media
maintained cohesive structure after incubation in media. Constructs
were imaged immediately following crosslinking step on day 0 and
before aerosol spray application on day 1 (FIG. 8). A bio-ink
comprised of fibroblasts is printed by continuous deposition to
create a dermal layer which is subsequently sprayed after a
maturation period of 24 hours by a cellular mixture of
keratinocytes and melanocytes to create an epidermal layer on top.
All constructs maintained a cohesive structure post printing with
continuous deposition and aerosol spray application. Constructs
contracted over time (FIG. 9) Skin tissues were cross sectioned
perpendicular to the plane of the transwell to show the base and
walls of the dermal cup structure. Sections were stained by H&E
(FIG. 10). Keratinocytes in the epidermal layer deposited by
aerosol spray were visualized by immunohistochemistry using
keratinocyte-specific marker cytokeratin 14 (CK14) (FIG. 10). CK14
is a marker for basal layer keratinocytes in normal human epidermal
skin and does not stain fibroblasts or melanocytes. Image shows
positive CK14 staining (keratinocytes) in red and dapi counter
staining nuclei in blue (fibroblasts). Positive staining can be
seen only on the apical surface of the construct where the aerosol
spray was applied. Staining shows an aerosol spray can successfully
apply a thin layer of cells to a surface. Current application shows
cells one layer thick. Positive CK14 staining is seen in constructs
at day 2, 4, and 6 (FIG. 11). Fibroblast tissue becomes flatter
over time as keratinocytes appear more rounded and potentially in
clusters (arrows). Differences in keratinocyte morphology suggest
potential differentiation over time. Keratinocytes were also
sprayed onto the surface of transwell membranes (FIG. 12). Cells
form similar groupings on transwell surface when compared to
morphology of keratinocytes sprayed onto dermal tissue constructs
day 8.
Example 2
Bioprinting Full Thickness Skin Tissue by Continuous Deposition
Using Dermal Bio-Ink Containing Gelatin and Epidermal Bio-Ink
Containing Cell Paste
Procedures
[0177] Bio-ink was generated by a cellular mixture of 100% primary
adult human dermal fibroblasts (HDFa) in 6% gelatin (Novogel.RTM.)
in a concentration of 150 million cells per milliliter.
Three-dimensional bio-ink constructs were printed by continuous
deposition using the Novogen Bioprinter platform in a 4 mm.times.4
mm.times.0.5 mm base sheet with a 1 mm wall bordering the top to
create a dermal structure resembling a cup. One tissue construct
was printed per transwell in a 6 well plate. The transwell printing
surface contained a polytetrafluoroethylene (PTFE) membrane coated
with equimolar mixture of types I and III collagen (bovine) with
pores 3 .mu.m in size. Epidermal cell paste containing a mixture of
95% primary adult human epidermal keratinocytes (HEKa) and 5%
primary adult human epidermal melanocytes (HEMa) was then printed
on top of the dermal bio-ink. Cell paste was measured post print at
90.5% viable by trypan exclusion assay. Cell number in deposited
epidermal layer was estimated at 160,000 cells by cell counting on
a Cell-0-Meter. Media was then added to the outer well of the
transwell in a volume of 2 ml. The media used for subsequent growth
and maintenance of the skin tissue was a 50:40:10 ratio of
HDFa:HEKa: HEMa media. The volume added was sufficient to collect
at the base of the printed structure but not to submerge the
structure. Media was changed 48 hours later and subsequently
changed daily after that. At days 2, 9, and 12, constructs were
either lysed for RNA analysis or fixed in 2% PFA for histological
analysis.
Results
[0178] H&E staining of skin tissues at day 12 shows a distinct
layered architecture (FIGS. 13, arrows, and 14). Fibroblasts in a
dermal layer are observed at the base (purple) and differentiated
keratinocytes in an epidermal layer (pink) on top. An unexpected
finding with this approach is the extent of the layered
architecture observed. In particular, there is a layer of cells
with distinct morphology can be observed at the interface (arrows).
This layer stains specifically for CK14, indicating that the
keratinocyte cells in the deposited paste have arranged into a
basal layer. Distinct layers of differentiated keratinocytes are
visualized by simultaneously staining for a basal cell marker CK5
and involucrin (IVL), a later stage differentiation marker of
granular and cornified keratinocytes. Similar to normal human skin,
differences in morphology are seen as basal cells appear to have a
distinct cuboidal morphology, while differentiated keratinocytes on
top appear flatter. The layered architecture also includes
CK10-positive spinous and granular keratinocytes in mid stages
differentiation (FIG. 15). Although previous print methods have
resulted in CK14 positive staining of the epidermal layer, the
observed pattern is widespread throughout the layer and
non-specific to a basal region at day 10. In the current approach,
what is unexpected is that the staining is limited to a defined
region at the base of the epidermal layer similar to native human
skin at day 12 (FIG. 16). Gene expression analysis supports
histological findings. Data shows an increase in epidermal
differentiation markers CK1, CK10, and especially late marker FLG
over time. Gene expression also shows that collagen 4 levels
increase over time, suggesting formation of a basement membrane.
Collagen I levels are maintained over the time course of the
experiment suggesting dermal layer remains viable (FIG. 17).
Example 3
Additional Example of Bioprinting Full Thickness Skin Tissue by
Continuous Deposition Using Dermal Bio-Ink Containing Gelatin and
Epidermal Bio-Ink Containing Cell Paste
Procedures
[0179] Bio-ink was generated by a cellular mixture of 100% primary
adult human dermal fibroblasts (HDFa) in 8% gelatin (Novogel.RTM.)
in a concentration of 100 million cells per milliliter. The cell:
gelatin ratio was altered to reduce the cellular density of the
dermal sheet to better mimic dermal tissue in native skin.
Three-dimensional bio-ink constructs were printed by continuous
deposition using the Novogen Bioprinter.RTM. platform in a 4
mm.times.4 mm.times.0.5 mm base sheet to create a dermal structure
resembling a sheet. One tissue construct was printed per
transwell-in a 6 well plate. The transwell printing surface
contained a polytetrafluoroethylene (PTFE)-membrane coated with
equimolar mixture of types I and III collagen (bovine) with pores 3
.mu.m in-size. Epidermal cell paste containing a mixture of 100%
primary neonatal human epidermal keratinocytes (HEKn) was then
printed on top of the dermal bio-ink. A separate but identical
epidermal paste structure was simultaneously deposited next to the
dermal sheet directly onto the transwell printing surface. This
structure was only comprised of epidermal keratinocyte paste and
contained no dermal tissue. Cell paste was measured post print at
87.1% viable by trypan exclusion assay. Cell number in deposited
epidermal layer was estimated at 60,000 cells by cell counting on a
Cell-O-Meter. Immediately following the print, constructs were
placed in 4.degree. C. for 10 minutes. This is a key step to harden
the Novogel.RTM., which helps to maintain the printed shape and
improve construct to construct uniformity. Cold media was then
added to the outer well of the transwell in a volume of 3 ml. The
media used for subsequent growth and maintenance of the skin tissue
was a 50:50 ratio of HDFa:HEKn media. The initial volume added was
sufficient to submerge the structure. All subsequent media changes
used warmed media (37.degree. C.) added to the outer well of the
transwell and not to the inner basket. Media was changed 48 hours
later and reduced to a volume of 1.5 ml per well to bring the
structure to an air-liquid interface (ALI). Media and subsequently
changed 48 hours after that (day 4) at a volume of 1.5 ml. On day
5, media was changed and further reduced to lml per well and
subsequently changed daily. At days 0 and 12, constructs were
either lysed for RNA analysis or fixed in 2% PFA for histological
analysis.
Results
[0180] Subsequent histological analysis to compare epidermal layer
patterning of paste that had been printed on top of a dermal sheet
versus directly onto the transwell surface yielded unexpected
findings (FIGS. 18 A and B). H&E staining of skin tissues at
day 12 shows a distinct layered architecture only in structures
with epidermal paste printed on top of a dermal layer (FIGS. 18 C
and D versus F and G, FIG. 19A). Fibroblasts in a dermal layer are
observed at the base (purple) and differentiated keratinocytes in
an epidermal layer (pink) on top. In particular, there is a layer
of cells with distinct morphology that can be observed at the
interface. Distinct layers of differentiated keratinocytes are
visualized by simultaneously staining for a basal cell marker CK5
(green) and involucrin (IVL, red), a later stage differentiation
marker of granular and cornified keratinocytes (FIG. 18 E versus H,
FIG. 19B). The distinct green layer indicates that the keratinocyte
cells in the deposited paste have arranged into a basal layer with
a layer of more differentiated IVL positive cells on top. Similar
to normal human skin, differences in morphology are seen as basal
cells that appear to have a distinct cuboidal morphology, while
differentiated keratinocytes on top appear flatter. Staining for
the proliferation marker PCNA (FIG. 19E, green) indicates that
proliferation is high in both dermal fibroblasts and basal layer
keratinocytes but not in differentiating keratinocytes. This
pattern is similar to that which is found in native skin. Staining
for apoptosis by TUNEL (FIG. 19F) also low showing very few
positive staining cells in either dermal or epidermal layer.
Collectively PCNA and TUNEL staining demonstrate that both dermal
and epidermal compartments of the full thickness tissue are viable
at day 12. Gene expression analysis supports histological findings.
Data shows an increase in mid epidermal differentiation markers
CK1, CK10, and later markers IVL, Loricrin, and at day 12 compared
to day 0. Gene expression also shows that collagen I and 4 levels
are maintained over the time course of the experiment, while
collagen 3 levels increase suggesting the dermal layer remains
viable and functional (FIG. 20). A number of surprising results
were determined from this; for example, that epidermal paste can
stratify into a distinct layered architecture. Current 3D skin
models rely on differentiation of a single keratinocyte monolayer
over an extended period of time to achieve this. Here we show that
stratification is possible to achieve with a paste. The thickness
of the paste is greater than a monolayer and shows that cells can
self-organize within the paste and differentiate as layers. Also,
we show that the keratinocyte paste printed directly onto the
transwell surface without the presence of dermal tissue did not
organize into stratified layers. Staining for the same
differentiation markers shows mixed expression with no defined
layers or distinct cell morphology. This unexpected finding
indicates that the dermal layer directs differentiation and/or
stratification of the epidermal keratinocytes, and that there is a
uniqueness to the combination of dermal and epidermal cells that is
not present in the epidermal cells alone. 3) The extent of the
layered architecture observed in the tissues comprised of both
epidermal and dermal cells including the staining of the
CK5-positive basal layer which is limited to a defined region at
the base of the epidermal layer similar to native human skin. The
layered architecture also includes a CK10 positive (FIG. 19C)
spinous and granular keratinocytes in mid stages differentiation
and with a morphologically distinct cornified layer of
keratinocytes visible by H&E and Trichrome staining above that
(FIGS. 19A and D respectively). A noteworthy advantage to this
approach is the appearance of the dermal layer. H&E staining
shows that the dermal fibroblasts do not form a thin sheet as in
earlier examples 1 and 2, but a thicker structure. Collagen
deposition, which is a key indicator of normal fibroblast function
in the dermis can be seen by both trichrome staining (blue color)
and by immunofluorescent staining for collagen 3 (red) in between
dermal cells (FIG. 19).
Example 4
Utilizing Bioprinted Full Thickness Skin Tissue in a Toxicology
Model with 1 Hour Exposure to Known Irritant 1% Triton
X-100.TM.
Procedures
[0181] Bio-ink was generated by a cellular mixture of 100% primary
adult human dermal fibroblasts (HDFa) in 8% gelatin (Novogel.RTM.
2.0) at a concentration of 100 million cells per milliliter. The
cell: gelatin ratio was altered to reduce the cellular density of
the dermal sheet to better mimic dermal tissue in native skin.
Three-dimensional bio-ink constructs were printed by continuous
deposition using the Novogen Bioprinter.RTM. platform in a 4
mm.times.4 mm.times.0.5 mm base to create a dermal structure
resembling a sheet. One tissue construct was printed per transwell
in a E-well plate. The transwell printing surface contained a
polytetrafluoroethylene (PTFE) membrane coated with equimolar
mixture of types I and III collagen (bovine) with pores 3 .mu.m in
size. Epidermal cell paste containing a mixture of 100% primary
neonatal human epidermal keratinocytes (HEKn) was then printed on
top of the dermal bio-ink. Cell paste was measured post print at
87.1% viable by trypan exclusion assay. Cell numbers in the
deposited epidermal layer was estimated at 60,000 cells by cell
counting on a Cell-O-Meter. Immediately following the print,
constructs were placed at 4.degree. C. for 10 minutes. Cold media
was then added to the outer well of the transwell in a volume of 3
ml. The media used for subsequent growth and maintenance of the
skin tissue was a 50:50 ratio of HDFa:HEKn media. The initial
volume added was sufficient to submerge the structure. All
subsequent media changes used warmed media (37.degree. C.) added to
the outer well of the transwell and not to the inner basket. Media
was changed 48 hours later and reduced to a volume of 1.5 ml per
well to bring the structure to an air-liquid interface (ALI). Media
and subsequently changed 48 hours after that (day 4) at a volume of
1.5 ml. On day 5, media was changed and further reduced to 1 ml per
well and subsequently changed daily. On day 10, skin tissues were
subject to a skin irritation test method using 1% Triton X 100. 1%
Triton X-100.TM. is a known irritant and reference chemical
currently used in other 3D skin models. Methods were based on OECD
guidelines 439 and 431 for applying human skin models to in vitro
skin irritation or corrosion, respectively. On day 10, conditioned
media was saved (designated time=0) and 1 ml of fresh media was
added to the outer well of the transwell. The printed skin tissue
was then treated with 20 .mu.l of PBS as a negative control or with
20 .mu.l of 1% Triton X-100.TM. as a positive control and known
irritant. Test substances were pipetted manually to the apical
surface of the tissue. Triton X-100.TM. was diluted to a 1% aqueous
solution in deionized water and sterile filtered through a 20
micron filter before use. Samples were incubated for 30 minutes at
37.degree. C. followed by 30 minutes at room temperature for a
total of 60 minutes. Media was saved (designated time=1 hour).
Samples were then washed extensively by PBS rinsing. Transwells
were then placed in a new 6-well plate with fresh media (1 ml)
added to the outer well then placed back into 37.degree. C.
overnight. The media was then changed at 24 hours and at 48 hours
post treatment. Total media collected included time points=-48, 0,
1, 24, and 48 hours. IL-1.alpha. production (R&D Systems) was
analyzed in a colorimetric assays per manufacturer's protocol.
Media was analyzed for lactate dehydrogenase as a marker for
cytotoxicity. IL-1.alpha. production was assessed as a
complementary endpoint to classic cytotoxicity testing to improve
predictability of irritants. Keratinocytes normally produce and
release inflammatory cytokine IL-1.alpha. in response to chemical
or physical stress. At 48 hours post treatment (day 12), tissues
were either lysed for RNA extraction and subsequent qPCR analysis,
or tested for viability by alamar blue assay then rinsed in PBS and
fixed with 2% PFA for histological analysis. Alamar blue (Life
Technologies) was added per manufacturer's instructions to media
and incubated with tissue constructs (1 ml) at 37.degree. C. for 3
hours. Viability was measured as fluorescence in the media by
conversion of resazurin to resorufin.
Results
[0182] The effect of Triton X-100 treatment on
viability/cytotoxicity was quantified by alamar blue assay, lactate
dehydrogenase (LDH) activity, and IL-1.alpha. production Skin
tissues treated with 1% Triton X 100 for 1 hour exhibited a roughly
2-fold induction in LDH activity compared to PBS control at 1 hour,
24 hours, and 48 hours point tested post treatment. In contrast,
PBS control is similar to initial baseline at 0 hours (FIG. 21A).
IL-1.alpha. activity also increased in response to Triton X
treatment in comparison to PBS control with the greatest difference
observed 1 hour post treatment. IL-1.alpha. levels remained high at
24 hours post treatment but returned to baseline at 48 hours (FIG.
21B). Comparison of tissues 48 hours post treatment shows an 80%
reduction in viability as compared to the PBS control (FIG. 18C).
H&E staining of skin tissues in the PBS control group at day 12
shows a distinct layered architecture (FIG. 22A). Fibroblasts in a
dermal layer are observed at the base (purple) and differentiated
keratinocytes in an epidermal layer (pink) on top. In particular,
there is a layer of cells with distinct morphology that can be
observed at the interface. Distinct layers of differentiated
keratinocytes are visualized by simultaneously staining for a basal
cell marker CK5 (green) and involucrin (IVL, red), a later stage
differentiation marker of granular and cornified keratinocytes
(FIG. 22B). The distinct green layer indicates that the
keratinocyte cells in the deposited paste have arranged into a
basal layer with a layer of more differentiated IVL positive cells
on top. Similar to normal human skin, differences in morphology are
seen as basal cells that appear to have a distinct cuboidal
morphology, while differentiated keratinocytes on top appear
flatter. The layered architecture also includes CK10-positive
spinous and granular keratinocytes in mid stages differentiation
(FIG. 22C). Collagen deposition, which is a key indicator of normal
fibroblast function in the dermis can be seen by both trichrome
staining (FIG. 22 D) (blue color) and by immunofluorescent staining
for collagen 3 (red) in between dermal cells (FIG. 22 E). Staining
for the proliferation marker PCNA (green) indicates that
proliferation is high in both dermal fibroblasts and basal layer
keratinocytes but not in differentiating keratinocytes. This
pattern is similar to that which is found in native skin. Staining
for apoptosis by TUNEL also low showing very few positive staining
cells in either dermal or epidermal layer (FIG. 22F). Collectively
PCNA and TUNEL staining demonstrate that both dermal and epidermal
compartments of the full thickness tissue are viable at day 12.
Tissues treated with Triton X appeared similar to PBS control
tissues macroscopically. Histological analysis, however revealed a
separation of the epidermal and dermal layers by H&E (FIG.
22G), suggesting a possible necrosis of the basement membrane
and/or basal keratinocyte layer. Although keratinocyte
differentiation markers CK5, IVL, and CK10 can be seen (FIGS. 22 H
and I), the CK5 positive basal keratinocyte layer is fragmented.
Collagen 3 staining is also compared to the PBS control indicating
a reduction in dermal fibroblast function (FIG. 22K) Viability is
also reduced and is consistent with biochemical data. This is
demonstrated by a reduction in proliferation evident by a less PCNA
positive staining in the dermal fibroblasts and lack of staining in
the basal layer. Combined with an increase in apoptosis as
demonstrated by TUNEL positive staining in the dermal layer (FIG.
22 L), histology suggests 1% Triton X is an irritant to bioprinted
skin. Gene expression analysis supports histological and
biochemical findings. Data shows an increase in epidermal
differentiation markers CK1, CK10, and late markers IVL, Loricrin,
and FLG over time in the PBS control group in comparison to tissue
at day 0. Gene expression also shows that collagen I and 4 levels
are maintained over the time course of the experiment, while
collagen 3 levels increase suggesting the dermal layer remains
viable (FIG. 23). Treatment with 1% Triton X dramatically reduces
expression of both epidermal and dermal markers 48 hours post
exposure (FIG. 23). CK1 and CK10 are reduced, suggesting a possible
effect to the spinous/granular layer of the epidermis. Collagen
production of types 1, 3, and 4 collagen is reduced, suggesting a
negative impact on the function of the dermal fibroblasts as well.
Inflammatory cytokine IL1a gene expression was also analyzed and
found to increase about 3 fold in comparison to the PBS control,
suggesting that 48 hours post treatment, keratinocytes are still
stressed by the exposure to Triton X (FIG. 24).
Example 5
Utilizing Bioprinted Full Thickness Skin Tissue in a Toxicology
Model with a 15 Minute Exposure to Known Irritants 5% SDS and 1%
Triton X when Miniaturized into a 24 Well Plate Format
Procedures
[0183] Bio-ink was generated by a cellular mixture of 100% primary
adult human dermal fibroblasts (HDFa) in 8% gelatin (Novogel.RTM.
2.0) in a concentration of 50 million cells per milliliter. The
cell: gelatin ratio was altered to further reduce the cellular
density of the dermal sheet to better mimic dermal tissue in native
skin. Three-dimensional bio-ink constructs were printed by
continuous deposition using the Novogen Bioprinter.RTM. platform in
a 2 mm.times.2 mm.times.0.5 mm base sheet to create a dermal
structure resembling a sheet. A smaller syringe needle was used to
achieve greater resolution at the reduced size. Instead of printing
one 0.5 mm sheet with a 500 .mu.m diameter syringe needle, two
layers of 0.25 mm sheets printed with a 250 .mu.m diameter needle.
One tissue construct was printed per transwell-in a 24 well plate.
The transwell printing surface contained a polytetrafluoroethylene
(PTFE)-membrane coated with equimolar mixture of types I and III
collagen (bovine) with pores 3 .mu.m in-size. Epidermal cell paste
containing a mixture of 100% primary neonatal human epidermal
keratinocytes (HEKn) was then printed on top of the dermal bio-ink.
Cell paste was measured post print at 96.0% viable by trypan
exclusion assay. Cell number in deposited epidermal layer was
estimated at 11,000 cells by cell counting on a Cell-O-Meter.
Immediately following the print, constructs were placed in
4.degree. C. for 10 minutes. This is a key step to harden the
Novogel.RTM., which helps to maintain the printed shape and improve
construct to construct uniformity. Cold media was then added to the
outer well of the transwell in a volume of 1 ml. The media used for
subsequent growth and maintenance of the skin tissue was a 50:50
ratio of HDFa:HEKn media. The initial volume added was sufficient
to submerge the structure. All subsequent media changes used warmed
media (37.degree. C.) added to the outer well of the transwell and
not to the inner basket. Media was changed 48 hours later and
reduced to a volume of 0.25 ml per well to bring the structure to
an air-liquid interface (ALI). Media and subsequently changed daily
Skin tissues were subject to a skin irritation test method using 1%
Triton X 100 and 5% SDS. 1% Triton X 100 and 5% SDS are known
irritants and reference chemicals currently used in other 3D skin
models. Methods were based on OECD guidelines 439 and 431 for
applying human skin models to in vitro skin irritation or
corrosion, respectively. On day 13, the media was changed. The
conditioned media was saved (designated time=0) and 1 ml of fresh
media was added to the outer well of the transwell. The printed
skin tissue was then treated with 5 .mu.l of PBS as a negative
control or with 5 .mu.l of 1% Triton X 100 or with 5 .mu.l of %%
SDS as a positive controls and known irritants. Test substances
were pipetted manually to the apical surface of the tissue. Both
Triton X 100 and SDS were diluted in an aqueous solution in
deionized water and sterile filtered through a 20 micron filter
before use. Samples were incubated for 15 minutes at room
temperature. Media was saved (designated time=15 minutes). Samples
were then washed extensively by PBS rinsing. Transwells were then
placed in a new 6 well plate with fresh media (0.25 ml) added to
the outer well then placed back into 37.degree. C. overnight. The
media was then changed again at 24 hours and 42 hours post
treatment. Total media collected included time points=0, 15
minutes, 24 hours, and 42 hours. IL-1.alpha. production (R&D
Systems) was analyzed in a colorimetric assays per manufacturer's
protocol. Media was analyzed for lactate dehydrogenase as a marker
for cytotoxicity. IL-1.alpha. production was assessed as a
complementary endpoint to classic cytotoxicity testing to improve
predictability of irritants. Keratinocytes produce and release
inflammatory cytokine IL-1.alpha. in response to chemical or
physical stress. At 42 hours post treatment (day 15), tissues were
either lysed for RNA extraction and subsequent qPCR analysis or
tested for viability by alamar blue assay then rinsed in PBS and
fixed with 2% PFA for histological analysis. Alamar blue (Life
Technologies) was added per manufacturer's instructions to media
and incubated with tissue constructs (0.25 ml) at 37.degree. C. for
3 hours. Viability was measured as fluorescence in the media by
conversion of resazurin to resorufin.
Results
[0184] Skin tissues exhibited an elevated LDH response to both SDS
and Triton X compared to PBS control tissues 42 hours post exposure
(FIG. 25A). The LDH activity in the SDS treated group was about 1.7
fold higher than PBS, while Triton X was about 1.4 fold higher
suggesting that 15 minute exposure with 5% SDS had a more
pronounced effect on construct viability than 1% Triton X. IL-1a
production was also induced by both irritants, however the SDS
treatment produced a much stronger response (FIG. 25B) consistent
with LDH activity. Tissues treated with both SDS and Triton X
exhibited a reduction in viability by alamar blue assay (FIG. 25C).
Tissues treated with SDS were only 2.45% viable compared to the PBS
control, while tissues treated with Triton X were 68.75% viable.
This data is consistent with the LDH activity and ILla production
suggesting that 5% SDS is a much stronger irritant than 1% Triton X
under these testing conditions. The difference in response to
Triton X in Example 4 compared to Example 5 may be due to the
shorter incubation time with the irritant.
[0185] 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 may be employed in
practicing the invention.
Example 6
Additional Example of Bioprinting Full Thickness Skin Tissue by
Continuous Deposition Using Dermal Bio-Ink Containing Gelatin,
Extracellular Matrix, and Epidermal Bio-Ink Containing Cell
Paste
Procedures
[0186] Bio-ink was generated by a cellular mixture of 100% primary
adult human dermal fibroblasts (HDFa) in 8% gelatin (Novogel.RTM.)
in a concentration of 100 million cells per milliliter.
Three-dimensional bio-ink constructs were printed by continuous
deposition using the Novogen Bioprinter.RTM. platform in a 3
mm.times.3 mm.times.0.5 mm base sheet. One tissue construct was
printed per transwell in a 12 well plate. The transwell printing
surface contained a polytetrafluoroethylene (PTFE) membrane coated
with equimolar mixture of types I and III collagen (bovine) with
pores 3 .mu.m in size. Extracellular matrix (ECM) containing a
mixture of type IV collagen, laminin, and heparin sulfate
proteoglycan was then printed on top of the dermal bio-ink at a
concentration of 120 micrograms per milliliter. Epidermal cell
paste containing 100% primary adult human epidermal keratinocytes
(HEKa) was then printed on top of the ECM. Cell paste was measured
post print at 94.4% viable by trypan exclusion assay. Cell number
in deposited epidermal layer was estimated at 14,000 cells by cell
counting on a Cell-O-Meter. Immediately following the print,
constructs were placed in 4.degree. C. for 10 minutes. Cold media
was then added to the outer well of the transwell in a volume of
0.5 ml. The media used for subsequent growth and maintenance of the
skin tissue was a 50:50 ratio of HDFa:HEKa media. The volume added
was sufficient to collect at the base of the printed structure but
not to submerge the structure. Media was again added after 90
minutes at a volume of 0.5 ml for a total of 1 ml in the well.
Media was changed 48 hours later at a volume of 1 ml. Media was
again changed after additional 24 hours (Day 3) at a volume of 0.5
ml to bring the structure to an air-liquid interface (ALI). Media
was subsequently changed at a volume of 0.5 ml every other day
after that. At day 10 constructs were fixed in 2% PFA for
histological analysis.
Results
[0187] H&E staining of skin tissues at day 10 shows a distinct
layered architecture (FIGS. 28 A and B). Fibroblasts in a dermal
layer are observed at the base and differentiated keratinocytes in
an epidermal layer on top. The distinct layered architecture of
differentiated keratinocytes is visualized by simultaneously
staining for a basal cell marker CK5 and differentiated marker
involucrin (IVL). Similar to normal human skin, differences in
morphology are seen as basal cells appear to have a distinct
cuboidal morphology, while differentiated keratinocytes on top
appear flatter (FIGS. 28 A and B). Although previous print methods
have resulted in CK5 positive staining of the basal keratinocyte
layer, the interface between the epidermal and dermal layers or
dermal-epidermal junction was not clearly demarcated. At the
interface between the basal layer of epidermal keratinocytes and
the dermal layer is the basement membrane. Types IV and VII
collagen are both specific markers of basement membrane formation.
In the current approach, what is unexpected is the expression and
extent of the organization of basement membrane markers types IV
and VII collagen. What is unexpected is that the staining patterns
of both markers is limited to a defined region, or line, precisely
at the base of the basal layer of epidermal keratinocytes at the
dermal-epidermal junction similar to native human skin (FIG. 28
E-H).
* * * * *