U.S. patent application number 11/932052 was filed with the patent office on 2008-05-08 for bioengineered tissue constructs and methods for producing and using thereof.
This patent application is currently assigned to Organogenesis, Inc.. Invention is credited to Michael P. Murphy, Vincent Ronfard.
Application Number | 20080108134 11/932052 |
Document ID | / |
Family ID | 26806787 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080108134 |
Kind Code |
A1 |
Murphy; Michael P. ; et
al. |
May 8, 2008 |
Bioengineered Tissue Constructs and Methods for Producing and Using
Thereof
Abstract
Cultured tissue constructs comprising cultured cells and
endogenously produced extracellular matrix components without the
requirement of exogenous matrix components or network support or
scaffold members. Some tissue constructs of the invention are
comprised of multiple cell layers or more than one cell type. The
tissue constructs of the invention have morphological features and
functions similar to tissues their cells are derived and their
strength makes them easily handleable. Preferred cultured tissue
constructs of the invention are prepared in defined media, that is,
without the addition of chemically undefined components.
Inventors: |
Murphy; Michael P.;
(Chelmsford, MA) ; Ronfard; Vincent; (Newton,
MA) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Assignee: |
Organogenesis, Inc.
Canton
MA
02021
|
Family ID: |
26806787 |
Appl. No.: |
11/932052 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09523809 |
Mar 13, 2000 |
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11932052 |
Oct 31, 2007 |
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PCT/US99/27505 |
Nov 19, 1999 |
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09523809 |
Mar 13, 2000 |
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09339632 |
Jun 24, 1999 |
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PCT/US99/27505 |
Nov 19, 1999 |
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60109247 |
Nov 19, 1998 |
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Current U.S.
Class: |
435/371 ;
435/366; 435/395 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 17/06 20180101; A61P 17/00 20180101; C12N 2502/094 20130101;
C12N 2500/90 20130101; C12N 5/0698 20130101; A61K 35/12 20130101;
A61P 17/02 20180101; C12N 2502/1323 20130101 |
Class at
Publication: |
435/371 ;
435/366; 435/395 |
International
Class: |
C12N 5/06 20060101
C12N005/06 |
Claims
1-30. (canceled)
31. A cultured tissue construct comprising fibroblasts cells grown
under conditions to produce a layer of extracellular matrix which
is synthesized and assembled by the fibroblast cells, with the
fibroblast cells surrounded by the synthesized extracellular matrix
layer, wherein the extracellular matrix is produced by the
fibroblast cells in the absence of exogenous matrix components or
synthetic members during the culturing conditions, and wherein the
conditions comprise a chemically defined medium.
32. The cultured tissue construct of claim 31, wherein fibroblasts
are derived from tissue selected from the group consisting of
neonate male foreskin, dermis, tendon, lung, urethra, umbilical
cord, corneal stroma, oral mucosa, intestine.
33. The cultured tissue construct of claim 31, wherein said
cultured cells are dermal fibroblasts.
34. The cultured tissue construct of claim 31, wherein said
cultured cells are from dermal papilla of hair follicles.
35. The cultured tissue construct of claim 31, wherein said layer
has cultured cells from dermal papilla of hair follicles are
localized on said layer.
36. The cultured tissue construct of claim 31, wherein said
cultured cells are derived from human tissue and are cultured in
medium containing no animal-derived components.
37. The cultured tissue construct of claim 31, wherein the cultured
cells are recombinant cells or genetically-engineered cells.
38. (canceled)
39. A method for producing a cultured tissue construct, comprising,
(a) seeding fibroblast cells capable of synthesizing an
extracellular matrix on a porous membrane in a culture vessel in a
first cell culture medium; (b) culturing the fibroblast cells of
step (a) in the first cell culture medium to between about 80% to
about 100% confluence on the porous membrane; (c) stimulating the
fibroblast cells to synthesize, secrete and organize extracellular
matrix components under culturing conditions in a second culture
medium; and, (d) continued culturing of the fibroblast cells until
the cells form a layer of synthesized extracellular matrix of at
least about 30 microns thick, with the cultured fibroblast cells
surrounded by the synthesized extracellular matrix layer, wherein
said extracellular matrix is produced by the cultured fibroblast
cells in the absence of exogenous matrix components or synthetic
members during the culturing conditions.
40. The method of claim 39, wherein either the first culture
medium, or the second culture medium, or both the first and second
culture mediums are chemically defined.
41. The method of claim 39, wherein the first and second culture
mediums contain no animal components.
42. The method of claim 39, wherein the second culture medium
comprises an ascorbate derivative.
43. The method of claim 42, wherein the ascorbate derivative is
sodium ascorbate, ascorbic acid, or L-ascorbic acid phosphate
magnesium salt n-hydrate.
44. The method of claim 39, wherein the second culture medium
comprises proline, glycine, or hydroxylproline.
45. The method of claim 42, wherein the second culture medium
further comprises proline, glycine, or hydroxylproline.
46. The method of claim 39, wherein the layer synthesized
extracellular matrix of at least about 60 microns thick.
47. The method of claim 46, wherein the layer synthesized
extracellular matrix of at least about 120 microns thick.
48. The method of claim 39, wherein the second culture medium
comprises a neutral polymer.
49. The method of claim 48, wherein the neutral polymer is
polyethylene glycol (PEG), dextran, polyvinylpyrrolidone (PVP).
50. The method of claim 39, wherein in step (a) the fibroblast
cells are seeded at a density between about 1.times.10.sup.5
cells/cm.sup.2 to about 6.6.times.10.sup.5 cells/cm.sup.2.
51. The method of claim 39, wherein the fibroblast cells are
derived from tissue selected from the group consisting of neonate
male foreskin, umbilical cord, dermis, tendon, lung, urethra,
corneal stroma, oral mucosa, and intestine.
52. A method for producing a bilayered cultured tissue construct,
comprising: (a) seeding fibroblast cells capable of synthesizing an
extracellular matrix on a porous membrane in a culture vessel in a
first cell culture medium; (b) culturing the fibroblast cells of
step (a) in the first cell culture medium to between about 80% to
about 100% confluence on the porous membrane; (c) stimulating the
fibroblast cells of step (a) to synthesize, secrete and organize
extracellular matrix components under culturing conditions in a
second culture medium; (d) continued culturing of the fibroblast
cells until the cells form a layer of synthesized extracellular
matrix of at least 30 microns thick, with the cultured fibroblast
cells surrounded by the synthesized extracellular matrix layer,
wherein said extracellular matrix is produced by the cultured
fibroblast cells in the absence of exogenous matrix components or
synthetic members during the culturing conditions; (e) seeding
epithelial cells to the top surface the synthesized extracellular
matrix of step (d), and, (f) stimulating the epithelial cells of
step (e) under culturing conditions to form a bilayered tissue
construct of an extracellular matrix, with the cultured fibroblast
cells surrounded by the synthesized extracellular matrix layer, and
a second layer of epithelial cells.
53. The method of claim 52, wherein either the first culture
medium, or the second culture medium, or both the first and second
culture mediums are chemically defined.
54. The method of claim 52, wherein the first and second culture
mediums contain no animal components.
55. The method of claim 52, wherein the second culture medium
comprises an ascorbate derivative.
56. The method of claim 55, wherein the ascorbate derivative is
sodium ascorbate, ascorbic acid, or L-ascorbic acid phosphate
magnesium salt n-hydrate.
57. The method of claim 52, wherein the second culture medium
comprises proline, glycine, or hydroxylproline.
58. The method of claim 55, wherein the second culture medium
further comprises proline, glycine, or hydroxylproline.
59. The method of claim 52, wherein the layer synthesized
extracellular matrix of at least about 60 microns thick.
60. The method of claim 59, wherein the layer synthesized
extracellular matrix of at least about 120 microns thick.
61. The method of claim 52, wherein the second culture medium
comprises a neutral polymer.
62. The method of claim 61, wherein the neutral polymer is
polyethylene glycol (PEG), dextran, polyvinylpyrrolidone (PVP).
63. The method of claim 52, wherein in step (a) the fibroblast
cells are seeded at a density between about 1.times.10.sup.5
cells/cm.sup.2 to about 6.6.times.10.sup.5 cells/cm.sup.2.
64. The method of claim 52, wherein the fibroblast cells are
derived from tissue selected from the group consisting of neonate
male foreskin, umbilical cord, dermis, tendon, lung, urethra,
corneal stroma, oral mucosa, and intestine.
65. The method of claim 52, wherein the epithelial cells are
selected from the group consisting of keratinocytes, corneal
epithelial cells, epithelial cells from oral mucosa, esophageal
epithelial cells, and uroepithelial cells.
66-105. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention is in the field of tissue engineering. This
invention is directed to an in vitro method for inducing cells to
produce an extracellular matrix. This living extracellular matrix,
which has tissue-like properties, can be used for testing or
clinical purposes.
BACKGROUND OF THE INVENTION
[0002] The field of tissue engineering combines bioengineering
methods with the principles of life sciences to understand the
structural and functional relationships in normal and pathological
mammalian tissues. The goal of tissue engineering is the
development and ultimate application of biological substitutes to
restore, maintain, or improve tissue functions. Thus, through
tissue engineering, it is possible to design and manufacture a
bioengineered tissue in a laboratory. Bioengineered tissues can
include cells that are usually associated with a native mammalian
or human tissues and synthetic or exogenous matrix scaffolds. The
new bioengineered tissue must be functional when grafted onto a
host, and be permanently incorporated within the host's body or
progressively bioremodeled by cells from the recipient host
patient. Fabrication of a tissue equivalent without a support
member or scaffold leads to scientific challenges in creating the
new bioengineered tissue.
SUMMARY OF THE INVENTION
[0003] The invention is directed to bioengineered tissue constructs
of cultured cells and endogenously produced extracellular matrix
components without the requirement of exogenous matrix components
or network support or scaffold members. The invention can thus
advantageously be made entirely from human cells, and human matrix
components produced by those cells, for example, when the
bioengineered tissue construct is designed for use in humans.
[0004] The invention is also directed to methods for producing
tissue constructs by stimulation of cells in culture, such as
fibroblasts, to produce extracellular matrix components without the
addition of either exogenous matrix components, network support, or
scaffold members.
[0005] The invention is also directed to methods for producing
tissue constructs by stimulation of cells in culture, such as
fibroblasts, to produce extracellular matrix components in a
defined medium system and/or without the use of undefined or
non-human-derived biological components, such as bovine serum or
organ extracts.
[0006] Further, this tissue construct can be made by serial
seedings of different cell types to produce a cultured tissue
construct that mimics the cell composition and tissue structures of
native tissues.
[0007] Still further, the tissue construct is produced and
self-assembled by cultured cells without the need for scaffold
support or the addition of exogenous extracellular matrix
components.
[0008] The strength characteristics of the tissue constructs make
it handleable for it to be easily and peelably removed from the
culture apparatus in which it is formed and directly transplanted
without the need for any support or carrier in clinical or testing
applications.
[0009] The tissue constructs of the invention are useful for
clinical purposes such as grafting to a patient with tissue or
organ defect, such as skin ulcer or wound, or for in vitro tissue
testing or animal grafting such as for safety testing or validation
of pharmaceutical, cosmetic, and chemical products.
DESCRIPTION OF THE FIGURES
[0010] FIG. 1 is a graph depicting the increase in collagen
concentration as determined by hydroxyproline assay as compared to
the cell number in the human neonatal foreskin cell derived dermal
construct described in Example 1.
[0011] FIG. 2 is a photomicrograph (objective 20.times.) of a
fixed, paraffin embedded, hematoxylin and eosin stained section of
a cell-matrix construct formed from cultured human dermal
fibroblasts in chemically defined medium at 21 days. The porous
membrane appears as a thin translucent band below the
construct.
[0012] FIG. 3 shows transmission electron microscope images of two
magnifications of a cell-matrix construct formed from cultured
human dermal fibroblasts in chemically defined medium at 21 days.
FIG. 3A is a 7600.times. magnification showing endogenous matrix
including alignment of collagen fibers between the fibroblasts.
FIG. 3B is a 19000.times. magnification of fully formed endogenous
collagen fibers demonstrating fibril arrangement and packing.
[0013] FIG. 4 is a photomicrograph (objective 20.times.) of a
fixed, paraffin embedded, hematoxylin and eosin stained section of
a cultured skin construct formed in chemically defined media in the
absence of exogenous matrix components comprising a cell-matrix
construct formed from cultured human dermal fibroblasts in
chemically defined medium with a multilayered, differentiated
epidermis formed from cultured human keratinocytes in chemically
defined medium.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Heretofore, current engineered living tissue constructs are
not completely cell assembled and must rely on either the addition
or incorporation of exogenous matrix components or synthetic
members for structure or support, or both.
[0015] The bioengineered tissue constructs descried herein exhibit
many of the native features of the tissue from which their cells
are derived. The tissue constructs thus produced can be used for
grafting to a patient or for in vitro testing.
[0016] One preferred embodiment is a cell-matrix construct
comprising a first cell type and endogenously produced
extracellular matrix wherein the first cell type is capable of
synthesizing and secreting extracellular matrix to produce the
cell-matrix construct.
[0017] Another preferred embodiment is a bilayer construct
comprising a first cell type and endogenously produced
extracellular matrix and a layer of cells of a second type disposed
thereon or within the cell-matrix construct formed by the first
cell type.
[0018] A more preferred embodiment is a cell-matrix construct
comprising fibroblasts, such as those derived from dermis, to form
a cultured dermal construct.
[0019] Another more preferred embodiment is a cell-matrix construct
comprising fibroblasts, such as those derived from dermis, to form
a cultured dermal construct with a layer of keratinocytes cultured
thereon to form an epidermal layer to result in a cultured bilayer
skin construct. The cultured skin constructs of the invention
express many physical morphological and biochemical features of
native skin.
[0020] In an even more preferred embodiment, the cell-matrix
construct is a tissue construct that is similar to the dermal layer
of skin, a human dermal construct, that is formed in a defined
system comprising human-derived cells utilizing no chemically
undefined components during its culture.
[0021] In the most preferred embodiment, the tissue constructs of
the invention are fabricated in a chemically defined system
comprising human-derived cells but no chemically undefined or
non-human biological components or cells.
[0022] One preferred embodiment of the invention comprises a
structural layer of at least one type of extracellular
matrix-producing cells and endogenously produced extracellular
matrix components, more simply termed "matrix", wherein the matrix
is completely cell-synthesized and assembled by culturing the
cells. This layer is herein termed a "cell-matrix construct" or a
"cell-matrix layer" because the cells secrete and contain
themselves within and through their matrix. The cultured tissue
constructs do not require, thus do not include, exogenous matrix
components, that is, matrix components not produced by the cultured
cells but introduced by other means. In a more preferred
embodiment, the cell-matrix construct produced by human dermal
fibroblasts is shown to have a predominant concentration of
collagen similar to native skin. As evidenced by electron
microscopy, the matrix is fibrous in nature comprising collagen
that exhibits the quarter-staggered 67 nm banding pattern, as well
as packing organization of fibrils and fibril bundles similar to
native collagen. Delayed reduction SDS-PAGE has detected the
presence of both type I and type III collagen in these constructs,
the predominant collagen types found in native human skin. Using
standard immunohistochemistry (IHC) techniques, the dermal
cell-matrix construct stains positively for decorin, a dermatan
sulfate proteoglycan known to be associated with collagen fibrils
and believed to regulate fibril diameter in vivo. Decorin can also
be visualized in the construct with TEM. The produced tissue also
stains positive for tenascin an extracellular matrix glycoprotein
found, for example, in mesenchyme or tissues under repair. Much
like tissue under repair in vivo, the tissue produced in culture
has been shown to increase its ratio of type I to type III collagen
as the matrix is formed. While not wishing to be bound by theory,
it is believed that the cells fill in the open space between them
quickly with a loose matrix analogous to granulation tissue
comprised of mostly type III collagen and fibronectin and then
remodel this loose matrix with a denser matrix comprised of mostly
type I collagen. The produced cell-matrix has been shown to contain
glycosaminoglycans (GAG), such as hyaluronic acid (HA);
fibronectin; proteoglycans besides decorin such as biglycan and
versican; and, a profile of sulfated glycosaminoglycans such as
di-hyaluronic acid; di-chondroitin-0-sulfate; di-chondroitin
4-sulfate; di-chondroitin-6-sulfate; di-chondroitin-4,6-sulfate;
di-chondroitin-4-sulfate-UA-2S; and di-chondroitin-6-sulfate-UA-2S.
These structural and biochemical features exhibit themselves as the
construct develops in culture and are distinctively evident when
the construct approaches its final form. The presence of these
components in fully formed cultured dermal cell-matrix construct
indicates that the construct has structural and biochemical
features approaching that of normal dermis.
[0023] While the aforementioned list is a list of biochemical and
structural features a cultured cell-matrix construct formed from
dermal fibroblasts, it should be recognized that cultured
cell-matrix constructs formed from other types of fibroblasts will
produce many of these features and others phenotypic for tissue
type from which they originated. In some cases, fibroblasts can be
induced to express non-phenotypic components by either chemical
exposure or contact, physical stresses, or by transgenic means.
Another preferred embodiment of the invention is a cell-matrix
layer having second layer of cells disposed thereon. The second
layer of cells is cultured on the cell-matrix layer to form a
bioengineered bilayered tissue construct. In a more preferred
embodiment, the cells of the second layer are of epithelial origin.
In the most preferred embodiment, the second layer comprises
cultured human keratinocytes that together with a first cell-matrix
layer, a cell-matrix construct formed from dermal fibroblasts and
endogenous matrix to form a dermal layer, comprise a living skin
construct. When fully formed, the epidermal layer is a
multilayered, stratified, and well-differentiated layer of
keratinocytes that exhibit a basal layer, a suprabasal layer, a
granular layer and a stratum corneum. The skin construct has a
well-developed basement membrane present at the dermal-epidermal
junction as exhibited by transmission electron microscopy (TEM).
The basement membrane appears thickest around hemidesmosomes,
marked by anchoring fibrils that are comprised of type VII
collagen, as visualized by TEM. The anchoring fibrils can seen
exiting from the basement membrane and entrapping the collagen
fibrils in the dermal layer. These anchoring fibrils, as well as
other basement membrane components, are secreted by keratinocytes.
It is also known that while keratinocytes are capable of secreting
basement membrane components on their own, a recognizable basement
membrane will not form in the absence of fibroblasts.
Immunohistochemical staining of the skin construct of the present
invention has also shown that laminin, a basement membrane protein
is present.
[0024] In a preferred method of the invention for forming a
cell-matrix construct, a first cell type, an extracellular
matrix-producing cell type, is seeded to a substrate, cultured, and
induced to synthesize and secrete an organized extracellular matrix
around them to form a cell-matrix construct. In another preferred
method of the invention, a surface of the cell-matrix construct is
seeded with cells of a second cell type and are cultured to form
bilayered tissue construct. In a more preferred method, a full
thickness skin construct having features similar to native human
skin is formed by culturing fibroblasts, such as human dermal
fibroblasts, under conditions sufficient to induce matrix synthesis
to form a cell-matrix of dermal cells and matrix, a dermal layer,
onto which human epithelial cells, such as keratinocytes, are
seeded and cultured under conditions sufficient to form a fully
differentiated stratified epidermal layer.
[0025] Therefore, one method of obtaining the tissue constructs of
the present invention comprises: [0026] (a) culturing at least one
extracellular matrix-producing cell type in the absence of
exogenous extracellular matrix components or a structural support
member, and, [0027] (b) stimulating the cells of step (a) to
synthesize, secrete, and organize extracellular matrix components
to form a tissue-construct comprised of cells and matrix
synthesized by those cells; wherein steps (a) and (b) may be done
simultaneously or consecutively.
[0028] To form a bilayer tissue construct comprising a cell-matrix
construct and a second cell layer thereon, the method additionally
comprises the step of: (c) culturing cells of a second type on a
surface of the formed tissue-construct to produce a bilayered
tissue construct.
[0029] An extracellular matrix-producing cell type for use in the
invention may be any cell type capable of producing and secreting
extracellular matrix components and organizing the extracellular
matrix components to form a cell-matrix construct. More than one
extracellular matrix-producing cell type may be cultured to form a
cell-matrix construct. Cells of different cell types or tissue
origins may be cultured together as a mixture to produce
complementary components and structures similar to those found in
native tissues. For example, the extracellular matrix-producing
cell type may have other cell types mixed with it to produce an
amount of extracellular matrix that is not normally produced by the
first cell type. Alternatively, the extracellular matrix-producing
cell type may also be mixed with other cell types that form
specialized tissue structures in the tissue but do not
substantially contribute to the overall formation of the matrix
aspect of the cell-matrix construct, such as in certain skin
constructs of the invention.
[0030] While any extracellular matrix-producing cell type may be
used in accordance with this invention, the preferred cell types
for use in this invention are derived from mesenchyme. More
preferred cell types are fibroblasts, stromal cells, and other
supporting connective tissue cells, most preferably human dermal
fibroblasts found in human dermis for the production of a human
dermal construct. Fibroblast cells, generally, produce a number of
extracellular matrix proteins, primarily collagen. There are
several types of collagens produced by fibroblasts, however, type I
collagen is the most prevalent in vivo. Human fibroblast cell
strains can be derived from a number of sources, including, but not
limited to neonate male foreskin, dermis, tendon, lung, umbilical
cords, cartilage, urethra, corneal stroma, oral mucosa, and
intestine. The human cells may include but need not be limited to
fibroblasts, but may include: smooth muscle cells, chondrocytes and
other connective tissue cells of mesenchymal origin. It is
preferred, but not required, that the origin of the
matrix-producing cell used in the production of a tissue construct
be derived from a tissue type that it is to resemble or mimic after
employing the culturing methods of the invention. For instance, in
the embodiment where a skin-construct is produced, the preferred
matrix-producing cell is a fibroblast, preferably of dermal origin.
In another preferred embodiment, fibroblasts isolated by
microdissection from the dermal papilla of hair follicles can be
used to produce the matrix alone or in association with other
fibroblasts. In the embodiment where a corneal-construct is
produced, the matrix-producing cell is derived from corneal stroma.
Cell donors may vary in development and age. Cells may be derived
from donor tissues of embryos, neonates, or older individuals
including adults. Embryonic progenitor cells such as mesenchymal
stem cells may be used in the invention and induced to
differentiate to develop into the desired tissue.
[0031] Although human cells are preferred for use in the invention,
the cells to be used in the method of the are not limited to cells
from human sources. Cells from other mammalian species including,
but not limed to, equine, canine, porcine, bovine, and ovine
sources; or rodent species such as mouse or rat may be used. In
addition, cells that are spontaneously, chemically or virally
transfected or recombinant cells or genetically engineered cells
may also be used in this invention. For those embodiments that
incorporate more than one cell type, chimeric mixtures of normal
cells from two or more sources; mixtures of normal and genetically
modified or transfected cells; or mixtures of cells of two or more
species or tissue sources may be used.
[0032] Recombinant or genetically-engineered cells may be used in
the production of the cell-matrix construct to create a tissue
construct that acts as a drug delivery graft for a patient needing
increased levels of natural cell products or treatment with a
therapeutic. The cells may produce and deliver to the patient via
the graft recombinant cell products, growth factors, hormones,
peptides or proteins for a continuous amount of time or as needed
when biologically, chemically, or thermally signaled due to the
conditions present in the patient. Either long or short-term gene
product expression is desirable, depending on the use indication of
the cultured tissue construct long term expression is desirable
when the cultured tissue construct is implanted to deliver
therapeutic products to a patient for an extended period of time.
Conversely, short term expression is desired in instances where the
cultured tissue construct is grafted to a patient having a wound
where the cells of the cultured tissue construct are to promote
normal or near-normal healing or to reduce scarification of the
wound site. Once the wound has healed, the gene products from the
cultured tissue construct are no longer needed or may no longer be
desired at the site. Cells may also be genetically engineered to
express proteins or different types of extracellular matrix
components which are either "normal" but expressed at high levels
or modified in some way to make a graft device comprising
extracellular matrix and living cells that is therapeutically
advantageous for improved wound healing, facilitated or directed
neovascularization, or minimized scar or keloid formation. These
procedures are generally known in the art, and are described in
Sambrook et al, Molecular Cloning A Laboratory Manual, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (1989), incorporated herein
by reference. All of the above-mentioned types of cells are
included within the definition of a "matrix-producing cell" as used
in this invention.
[0033] The predominant major extracellular matrix component
produced by fibroblasts is fibrillar collagen, particularly
collagen type I. Fibrillar collagen is a key component in the
cell-matrix structure; however, this invention is not to be limited
to matrices of only this protein or protein type. For instance,
other collagens, both fibrillar and non-fibrillar collagen from the
collagen family such as collagen types II, III, IV, V, VI, VII,
VII, IX, X, XI, XII, XIII, XV, XV, XVI, XVII, XVIII, XXI, may be
produced by use of the appropriate cell type. Similarly, other
matrix proteins which can be produced and deposited using the
current method include, but are not limited to elastin;
proteoglycan such as decorin or biglycan; or glycoproteins such as
tenascin; vitronectin; fibronectin; laminin, thrombospondin I, and
glycosaminoglycan (GAG) such as hyaluronic acid (HA).
[0034] The matrix-producing cell is cultured in a vessel suitable
for animal cell or tissue culture, such as a culture dish, flask,
or roller-bottle, which allows for the formation of a
three-dimensional tissue-like structure. Suitable cell growth
surfaces on which the cells can be grown can be any biologically
compatible material to which the cells can adhere and provide an
anchoring means for the cell-matrix construct to form. Materials
such as glass; stainless steel; polymers, including polycarbonate,
polystyrene, polyvinyl chloride, polyvinylidene,
polydimethylsiloxane, fluoropolymers, and fluorinated ethylene
propylene; and silicon substrates, including fused silica,
polysilicon, or silicon crystals may be used as a cell growth
surfaces. The cell growth surface material may be chemically
treated or modified, electrostatically charged, or coated with
biologicals such as poly-1-lysine or peptides. An example of a
peptide coating is RGD peptide.
[0035] While the tissue construct of the invention may be grown on
a solid cell growth surface, a cell growth surface with pores that
communicate both top and bottom surfaces of the membrane to allow
bilateral contact of the medium to the developing tissue construct
or for contact from only below the culture is preferred. Bilateral
contact allows medium to contact both the top and bottom surfaces
of the developing construct for maximal surface area exposure to
the nutrients contained in the medium. Medium may also contact only
the bottom of the forming cultured tissue construct so that the top
surface may be exposed to air, as in the development of a cultured
skin construct. The preferred culture vessel is one that utilizes a
carrier insert, a culture-treated permeable member such as a porous
membrane that is suspended in the culture vessel containing medium.
Typically, the membrane is secured to one end of a tubular member
or framework that is inserted within and interfaces with a base,
such as a petri or culture dish that can be covered with a lid.
Culture vessels incorporating a carrier insert with a porous
membrane are known in the art and are preferred for carrying out
the invention and are described in a number U.S. patents in the
field, some of which have been made commercially available,
including for instance: U.S. Pat. Nos. 5,766,937, 5,466,602,
5,366,893, 5,358,871, 5,215,920, 5,026,649, 4,871,674, 4,608,342,
the disclosures of which are incorporated herein. When these types
of culture vessels are employed, the tissue-construct is produced
on one surface of the membrane, preferably the top, upwardly facing
surface and the culture is contacted by cell media on both top and
bottom surfaces. The pores in the growth surface allow for the
passage of culture media for providing nutrients to the underside
of the culture through the membrane, thus allowing the cells to be
fed bilaterally or solely from the bottom side. A preferred pore
size is one that is small enough that it does not allow for the
growth of cells through the membrane, yet large enough to allow for
free passage of nutrients contained in culture medium to the bottom
surface of the cell-matrix construct, such as by capillary action.
Preferred pore sizes are about less than 3 microns but range
between about 0.1 microns to about 3 microns, more preferably
between about 0.2 microns to about 1 micron and most preferably
about 0.4 micron to about 0.6 micron sized pores are employed. In
the case of human dermal fibroblasts, the most preferred material
is polycarbonate having a pore size is between about 0.4 to about
0.6 microns. The maximum pore size depends not only on the size of
the cell but also the ability of the cell to alter its shape and
pass through the membrane. It is important that the tissue-like
construct adheres to the surface but does not incorporate or
envelop the substrate so it is removable from it such as by peeling
with minimal force. The size and shape of the tissue construct
formed is dictated by the size of the vessel surface or membrane on
which it grown. Substrates may be round or angular or shaped with
rounded corner angles, or irregularly shaped. Substrates may also
be flat or contoured as a mold to produce a shaped construct to
interface with a wound or mimic the physical structure of native
tissue. To account for greater surface areas of the growth
substrate, proportionally more cells are seeded to the surface and
a greater volume of media is needed to sufficiently bathe and
nourish the cells. When the tissue construct is finally formed,
whether it is a single layer cell-matrix construct or a bilayer
construct, it is removed by peeling from the membrane substrate
before grafting to a patient.
[0036] The cultured tissue constructs of the invention do not rely
on synthetic or bioresorbable members for, such as a mesh member
for the formation of the tissue constructs. The mesh member is
organized as a woven, a knit, or a felt material. In systems where
a mesh member is employed, the cells are cultured on the mesh
member and growing on either side and within the interstices of the
mesh to envelop and incorporate the mesh within the cultured tissue
construct. The final construct formed by methods that incorporate
such a mesh rely on it for physical support and for bulk Examples
of cultures tissue constructs that rely on synthetic mesh members
are found in U.S. Pat. Nos. 5,580,781, 5,443,950, 5,266,480,
5,032,508, 4,963,489 to Naughton, et al.
[0037] The system for the production of the cell-matrix layer may
be either static or may employ a perfusion means to the culture
media. In the static system, the culture medium is still and
relatively motionless as contrasted to the perfusion system where
the medium is in motion. The perfusion of medium affects the
viability of the cells and augments the development of the matrix
layer. Perfusion means include, but are not limited to: using a
magnetic stirbar or motorized impeller in the culture dish
subjacent (below) or adjacent to the substrate carrier containing
the culture membrane to stir the medium; pumping medium within or
through the culture dish or chamber, gently agitating the culture
dish on a shaking or rotating platform; or rolling, if produced in
a roller bottle. Other perfusion means can be determined by one
skilled in the art for use in the method of the invention.
[0038] Culture media formulations suitable for use in the present
invention are selected based on the cell types to be cultured and
the tissue structure to be produced. The culture medium that is
used and the specific culturing conditions needed to promote cell
growth, matrix synthesis, and viability will depend on the type of
cell being grown.
[0039] In some instances, such as in the fabrication of
bioengineered bilayer skin constructs of the present invention, the
media composition varies with each stage of fabrication as
different supplementation is necessary for different purposes. In a
preferred method, the cell-matrix layer is formed under defined
conditions, that is, cultured in chemically defined media. In
another preferred method, a tissue construct comprises a
cell-matrix layer provided with a second layer of cells disposed
and cultured thereon wherein both cell types are cultured in a
defined culture media system. Alternatively, the tissue construct
comprises a cell-matrix layer fabricated under defined media
conditions and a second layer formed thereon under undefined media
conditions. In the converse, the tissue construct comprises a
cell-matrix layer may be fabricated under undefined media
conditions and the second layer formed thereon under defined media
conditions.
[0040] The use of chemically defined culture media is preferred,
that is, media free of undefined animal organ or tissue extracts,
for example, serum, pituitary extract, hypothalamic extract,
placental extract, or embryonic extract or proteins and factors
secreted by feeder cells. In a most preferred embodiment, the media
is free of undefined components and defined biological components
derived from non-human sources. Although the addition of undefined
components is not preferred, they may be used in accordance with
the disclosed methods at any point in culture in order to fabricate
successfully a tissue construct. When the invention is carried out
utilizing screened human cells cultured using chemically defined
components derived from no non-human animal sources, the resultant
tissue construct is a defined human tissue construct. Synthetic
functional equivalents may also be added to supplement chemically
defined media within the purview of the definition of chemically
defined for use in the most preferred fabrication method.
Generally, one of skill in the art of cell culture will be able to
determine suitable natural human, human recombinant, or synthetic
equivalents to commonly known animal components to supplement the
culture media of the invention without undue investigation or
experimentation. The advantages in using such a construct in the
clinic is that the concern of adventitious animal or cross-species
virus contamination and infection is diminished. In the testing
scenario, the advantages of a chemically defined construct is that
when tested, there is no chance of the results being confounded due
to the presence of the undefined components.
[0041] Culture medium is comprised of a nutrient base usually
further supplemented with other components. The skilled artisan can
determine appropriate nutrient bases in the art of animal cell
culture with reasonable expectations for successfully producing a
tissue construct of the invention. Many commercially available
nutrient sources are useful on the practice of the present
invention. These include commercially available nutrient sources
which supply inorganic salts, an energy source, amino acids, and
B-vitamins such as Dulbecco's Modified Eagle's Medium (DMEM);
Minimal Essential Medium (MEM); M199; RPMI 1640; Iscove's Modified
Dulbecco's Medium (EDMEM). Minimal Essential Medium (MEM) and M199
require additional supplementation with phospholipid precursors and
non essential amino acids. Commercially available vitamin-rich
mixtures that supply additional amino acids, nucleic acids, enzyme
cofactors, phospholipid precursors, and inorganic salts include
Ham's F12, Ham's F-10, NCTC 109, and NCTC 135. Albeit in varying
concentrations, all basal media provide a basic nutrient source for
cells in the form of glucose, ammo acids, vitamins, and inorganic
ions, together with other basic media components. The most
preferred base medium of the invention comprises a nutrient base of
either calcium-free or low calcium Dulbecco's Modified Eagle's
Medium (DMEM), or, alternatively, DMEM and Ham's F12 between a
3-to-1 ratio to a 1-to-3 ratio, respectively.
[0042] The base medium is supplemented with components such as
amino acids, growth factors, and hormones. Defined culture media
for the culture of cells of the invention are described in U.S.
Pat. No. 5,712,163 to Parenteau and in International PCT
Publication No. WO 95/31473, the disclosures of which are
incorporated herein by reference. Other media are known in the art
such as those disclosed in Ham and McKeehan, Methods in Enzymology,
58:44-93 (1979), or for other appropriate chemically defined media,
in Bottenstein et al., Methods in Enzymology, 58:94-109 (1979). In
the preferred embodiment, the base medium is supplemented with the
following components known to the skilled artisan in animal cell
culture: insulin, transferrin, triiodothyronine (T3), and either or
both ethanolamine and o-phosphoryl-ethanolamine, wherein
concentrations and substitutions for the supplements may be
determined by the skilled artisan.
[0043] Insulin is a polypeptide hormone that promotes the uptake of
glucose and amino acids to provide long term benefits over multiple
passages. Supplementation of insulin or insulin-like growth factor
(IGF) is necessary for long term culture as there will be eventual
depletion of the cells' ability to uptake glucose and amino acids
and possible degradation of the cell phenotype. Insulin may be
derived from either animal, for example bovine, human sources, or
by recombinant means as human recombinant insulin. Therefore, a
human insulin would qualify as a chemically defined component not
derived from a non-human biological source. Insulin supplementation
is advisable for serial cultivation and is provided to the media at
a wide range of concentrations. A preferred concentration range is
between about 0.1 .mu.g/ml to about 500 .mu.g/ml more preferably at
about 5 .mu.g/ml to about 400 .mu.g/ml, and most preferably at
about 375 .mu.g/ml. Appropriate concentrations for the
supplementation of insulin-like growth factor, such as IGF-1 or
IGF-2, may be easily determined by one of skill in the art for the
cell types chosen for culture.
[0044] Transferrin is in the medium for iron transport regulation.
Iron is an essential trace element found in serum. As iron can be
toxic to cells in its free form, in serum it is supplied to cells
bound to transferrin at a concentration range of preferably between
about 0.05 to about 50 .mu.g/ml, more preferably at about 5
.mu.g/ml.
[0045] Triiodothyronine (T3) is a basic component and is the active
form of thyroid hormone that is included in the medium to maintain
rates of cell metabolism. Triiodothyronine is supplemented to the
medium at a concentration range between about 0 to about 400 .mu.M,
more preferably between about 2 to about 200 .rho.M and most
preferably at about 20 .rho.M.
[0046] Either or both ethanolamine and o-phosphoryl-ethanolamine,
which are phospholipids, are added whose function is an important
precursor in the inositol pathway and fatty acid metabolism.
Supplementation of lipids that are normally found in serum is
necessary in a serum-free medium. Ethanolamine and
o-phosphoryl-anolamine are provided to media at a concentration
range between about 10.sup.-6 to about 10.sup.-2 M, more preferably
at about 1.times.10.sup.-4 M.
[0047] Throughout the culture duration, the base medium is
additionally supplemented with other components to induce synthesis
or differentiation or to improve cell growth such as
hydrocortisone, selenium, and I-glutamine.
[0048] Hydrocortisone has been shown in keratinocyte culture to
promote keratinocyte phenotype and therefore enhance differentiated
characteristics such as involucrin and keratinocyte
transglutaminase content (Rubin et al., J. Cell Physiol.,
138:208-214 (1986)). Therefore, hydrocortisone is a desirable
additive in instances where these characteristics are beneficial
such as in the formation of keratinocyte sheet grafts or skin
constructs. Hydrocortisone may be provided at a concentration range
of about 0.01 ug/ml to about 4.0 .mu.g/ml, most preferably between
about 0.4 .mu.g/ml to 16 ug/ml.
[0049] Selenium is added to serum-free media to resupplement the
trace elements of selenium normally provided by serum. Selenium may
be provided at a concentration range of about 10.sup.-9 M to about
10.sup.-7 M; most preferably at about 5.3.times.10.sup.-8 M.
[0050] The amino acid L-glutamine is present in some nutrient bases
and may be added in cases where there is none or insufficient
amounts present L-glutamine may also be provided in stable form
such as that sold under the mark, GlutaMAX-1.TM. (Gibco BRL, Grand
Island, N.Y.). GlutaMAX-1.TM. is the stable dipeptide form of
L-alanyl-glutamine and may be used interchangeably with L-glutamine
and is provided in equimolar concentrations as a substitute to
L-glutamine. The dipeptide provides stability to L-glutamine from
degradation over time in storage and during incubation that can
lead to uncertainty in the effective concentration of L-glutamine
in medium. Typically, the base medium is supplemented with
preferably between about 1 mM to about 6 mM, more preferably
between about 2 mM to about 5 mM and most preferably 4 mM
L-glutamine or GlutaMAX-1.TM..
[0051] Growth factors such as epidermal growth factor (EGF) may
also be added to the medium to aid in the establishment of the
cultures through cell scale-up and seeding. EGF in native form or
recombinant form may be used. Human forms, native or recombinant,
of EGF are preferred for use in the medium when fabricating a skin
equivalent containing no non-human biological components. EGF is an
optional component and may be provided at a concentration between
about 1 to 15 ng/mL, more preferably between about 5 to 10
ng/mL.
[0052] The medium described above is typically prepared as set
forth below. However, it should be understood that the components
of the present invention may be prepared and assembled using
conventional methodology compatible with their physical properties.
It is well known in the art to substitute certain components with
an appropriate analogous or functionally equivalent acting agent
for the purposes of availability or economy and arrive at a similar
result. Naturally occurring growth factors may be substituted with
recombinant or synthetic growth factors that have similar qualities
and results when used in the performance of the invention.
[0053] Media in accordance with the present invention are sterile.
Sterile components are bought sterile or rendered sterile by
conventional procedures, such as filtration, after preparation.
Proper aseptic procedures were used throughout the following
Examples. DMEM and F-12 are first combined and the individual
components are then added to complete the medium. Stock solutions
of all components can be stored at -20.degree. C., with the
exception of nutrient source that can be stored at 4.degree. C. All
stock solutions are prepared at 500.times. final concentrations
listed above. A stock solution of insulin, transferrin and
triiodothyronine (all from Sigma) is prepared as follows:
triiodothyronine is initially dissolved in absolute ethanol in 1N
hydrochloric acid (HCl) at a 2:1 ratio. Insulin is dissolved in
dilute HCl (approximately 0.1N) and transferrin is dissolved in
water. The three are then mixed and diluted in water to a
500.times. concentration. Ethanolamine and
o-phosphoryl-ethanolamine are dissolved in water to 500.times.
concentration and are filter sterilized. Progesterone is dissolved
in absolute ethanol and diluted with water. Hydrocortisone is
dissolved in absolute ethanol and diluted in phosphate buffered
saline (PBS). Selenium is dissolved in water to 500.times.
concentration and filter sterilized. EGF is purchased sterile and
is dissolved in PBS. Adenine is difficult to dissolve but may be
dissolved by any number of methods known to those skilled in the
art. Serum albumin may be added to certain components in order to
stabilize them in solution and are presently derived from either
human or animal sources. For example, human serum albumin (HSA) or
bovine serum albumin (BSA) may be added for prolonged storage to
maintain the activity of the progesterone and EGF stock solutions.
The medium can be either used immediately after preparation or,
stored at 4.degree. C. If stored, EGF should not be added until the
time of use.
[0054] In order to form the cell-matrix layer by the culture of
matrix-producing cells, the medium is supplemented with additional
agents that promote matrix synthesis and deposition by the cells.
These supplemental agents are cell-compatible, defined to a high
degree of purity and are free of contaminants. The medium used to
produce the cell-matrix layer is termed "matrix production
medium".
[0055] To prepare the matrix production medium, the base medium is
supplemented with an ascorbate derivative such as sodium ascorbate,
ascorbic acid, or one of its more chemically stable derivatives
such as L-ascorbic acid phosphate magnesium salt n-hydrate.
Ascorbate is added to promote hydroxylation of proline and
secretion of procollagen, a soluble precursor to deposited collagen
molecules. Ascorbate has also been shown to be an important
cofactor for post-translational processing of other enzymes as well
as an upregulator of type I and type III collagen synthesis.
[0056] While not wishing to be bound by theory, supplementing the
medium with amino acids involved in protein synthesis conserves
cellular energy by not requiring the cells produce the amino acids
themselves. The addition of proline and glycine is preferred as
they, as well as the hydroxylated form of proline, hydroxyproline,
are basic amino acids that make up the structure of collagen.
[0057] While not required, the matrix-production medium is
optionally supplemented with a neutral polymer. The cell-matrix
constructs of the invention may be produced without a neutral
polymer, but again not wishing to be bound by theory, its presence
in the matrix production medium may collagen processing and
deposition more consistently between samples. One preferred neutral
polymer is polyethylene glycol (PEG), which has been shown to
promote in vitro processing of the soluble precursor procollagen
produced by the cultured cells to matrix deposited collagen. Tissue
culture grade PEG within the range between about 1000 to about 4000
MW (molecular weight), more preferably between about 3400 to about
3700 MW is preferred in the media of the invention. Preferred PEG
concentrations are for use in the method may be at concentrations
at about 5% w/v or less, preferably about 0.01% w/v to about 0.5%
w/v, more preferably between about 0.025% w/v to about 0.2% w/v,
most preferably about 0.05% w/v. Other culture grade neutral
polymers such dextran, preferably dextran T-40, or
polyvinylpyrrolidone (PVP), preferably in the range of
30,000-40,000 MW, may also be used at concentrations at about 5%
w/v or less, preferably between about 0.01% w/v to about 0.5% w/v,
more preferably between about 0.025% w/v to about 0.2% w/v, most
preferably about 0.05% w/v. Other cell culture grade and
cell-compatible agents that enhance collagen processing and
deposition may be ascertained by the skilled routineer in the art
of mamma cell culture.
[0058] When the cell producing cells are confluent, and the culture
medium is supplemented with components that assist in matrix
synthesis, secretion, or organization, the cells are said to be
stimulated to form a tissue-construct comprised of cells and matrix
synthesized by those cells.
[0059] Therefore, a preferred matrix production medium formulation
comprises: a base 3:1 mixture of Dulbecco's Modified Eagle's Medium
(DMEM) (high glucose formulation, without L, glutamine) and Hams
F-12 medium supplemented with either 4 mM L-glutamine or
equivalent, 5 ng/ml epidermal growth factor, 0.4 .mu.g/ml
hydrocortisone, 1.times.10.sup.-4 M ethanolamine, 1.times.10.sup.-4
M o-phosphoryl-ethanolamine, 5 .mu.g/ml insulin, 5 .mu.g/ml
transferrin, 20 .rho.M triiodothyronine, 6.78 ng/ml selenium, 50
ng/ml L-ascorbic acid, 0.2 .mu.g/ml L-proline, and 0.1 .mu.g/ml
glycine. To the production medium, other pharmacological agents may
be added to the culture to alter the nature, amount, or type of the
extracellular matrix secreted. These agents may include polypeptide
growth factors, transcription factors or inorganic salts to
up-regulate collagen transcription Examples of polypeptide growth
factors include transforming growth factor-beta 1 (TGF-.beta.1) and
tissue-plasminogen activator (TPA), both of which are known to
upregulate collagen synthesis. Raghow et al., Journal of Clinical
Investigation, 79:1285-1288 (1987); Pardes et al., Journal of
Investigative Dermatology, 100:549 (1993). An example of an
inorganic salt that stimulates collagen production is cerium.
Shivakumar et al., Journal of Molecular and Cellular Cardiology
24:775-780 (1992).
[0060] The cultures are maintained in an incubator to ensure
sufficient environmental conditions of controlled temperature,
humidity, and gas mixture for the culture of cells. Preferred
conditions are between about 34.degree. C. to about 38.degree. C.,
more preferably 37.+-.1.degree. C. with an atmosphere between about
5-10.+-.1% CO.sub.2 and a relative humidity (Rh) between about
80-90%.
[0061] In the preferred embodiment, the cell-matrix construct is a
dermal construct formed of dermal fibroblasts and their secreted
matrix. Preferably, human dermal fibroblasts are used, derived as
primary cells from dermis or more preferably from serially passaged
or subcultured from established cell stocks or banks that have been
screened against viral and bacterial contamination and tested for
purity. Cells are cultured under sufficient conditions in growth
medium to cause them to proliferate to an appropriate number for
seeding the cells to the culture substrate on which to form a
cell-matrix construct. Alternatively, cells from frozen cell stocks
may be seeded directly to the culture substrate.
[0062] Once sufficient cell numbers have been obtained, cells are
harvested and seeded onto a suitable culture surface and cultured
under appropriate growth conditions to form a confluent sheet of
cells. In the preferred embodiment, the cells are seeded on a
porous membrane that is submerged to allow medium contact from
below the culture through the pores and directly above. Preferably,
cells are suspended in either base or growth media and are seeded
on the cell culture surface at a density between about
1.times.10.sup.5 cells/cm.sup.2 to about 6.6.times.10.sup.5
cells/cm.sup.2, more preferably between about 3.times.10.sup.5
cells/cm.sup.2 to about 6.6.times.10.sup.5 cells/cm.sup.2 and most
preferably at about 6.6.times.10.sup.5 cells/cm.sup.2 (cells per
square centimeter area of the surface). Cultures are cultured in
growth medium to establish the culture and are cultured to between
about 80% to 100% confluence at which time they are induced
chemically by changing the medium to matrix production medium in
order to upregulate the synthesis and secretion of extracellular
matrix. In an alternate method, cells are seeded directly in
production media to emnate the need to change from the basic media
to the production media but it is a method that requires higher
seeding densities.
[0063] During the culture, fibroblasts organize the secreted matrix
molecules to form a three dimensional tissue-like structure but do
not exhibit significant contractile forces to cause the forming
cell-matrix construct to contract and peel itself from the culture
substrate. Media exchanges are made every two to three days with
fresh matrix production medium and with time, the secreted matrix
increases in thickness and organization. The time necessary for
creating a cell-matrix construct is dependent on the ability of the
initial seeding density, the cell type, the age of the cell line,
and the ability of the cell line to synthesize and secrete matrix.
When fully formed, the constructs of the invention have bulk
thickness due to the fibrous matrix produced and organized by the
cells; they are not ordinary confluent or overly confluent cell
cultures where the cells may be loosely adherent to each other. The
fibrous quality gives the constructs cohesive tissue-like
properties unlike ordinary cultures because they resist physical
damage, such as tearing or cracking, with routine handling in a
clinical setting. In the fabrication of a cultured dermal
construct, the cells win form an organized matrix around themselves
on the cell culture surface preferably at least about 30 microns in
thickness or more, more preferably between about 60 to about 120
microns thick across the surface of the membrane; however,
thicknesses have been obtained in excess of 120 microns and are
suitable for use in testing or clinical applications where such
greater thicknesses are needed.
[0064] In a more preferred method, an epithelial cell layer is
applied to one surface, preferably the top, upwardly facing surface
of the cell-matrix construct. To the cell-matrix construct,
epithelial cells may be seeded and cultured thereon to form a
multilayer tissue construct. In the most to preferred method,
keratinocytes derived from skin are grown on the cell construct to
form a skin construct. In other preferred embodiments, corneal
epithelial cells, also termed corneal keratinocytes, may be seeded
on the cell-matrix construct to form a corneal construct.
Epithelial cells from the oral mucosa may be grown on the
cell-matrix construct to form a construct of oral mucosa.
Epithelial cells from esophagus may be seeded on the cell-matrix
construct to form a construct of esophageal tissue. Uroepithelial
cells from the urogenital tract may be seeded on the cell-matrix
construct to form a construct of uroepithelium. Other cells of
epithelial origin may be selected to form a construct of tissue
from which those cells were derived.
[0065] Methods for providing epidermal cells to a dermal substrate,
and methods for their culture, including induction of
differentiation and cornification to form a differentiated
keratinocyte layer are known in the art and are described in U.S.
Pat. No. 5,712,163 to Parenteau, et al. and in U.S. Pat. No.
5,536,656 to Kemp, et al., the contents of which are incorporated
herein by reference. Typically to perform the epidermalization of
the cell-matrix construct, keratinocytes are seeded to the
cell-matrix construct and cultured thereon until the layer is about
one to three cell layers thick. The keratinocytes are then induced
to differentiate to form a multilayer epidermis and are then
induced to cornify to form a stratum corneum.
[0066] In the method of forming a differentiated epidermal layer,
subcultured keratinocytes are taken from the cell stock and their
cell numbers are expanded. When an necessary number of cells have
been obtained, they are released from the culture substrate,
suspended, counted, diluted and then seeded to the top surface of
the cell-matrix construct at a density between about
4.5.times.10.sup.3 cells/cm.sup.2 to about 5.0.times.10.sup.5
cells/cm.sup.2, more preferably between about 1.0.times.10.sup.4
cells/cm.sup.2 to about 1.0.times.10.sup.5 cells/cm.sup.2, and most
preferably at about 4.5.times.10.sup.4 cells/cm.sup.2. The
constructs are then incubated for between about 60 to about 90
minutes at 37.+-.1.degree. C., 10% CO.sub.2 to allow the
keratinocytes to attach. After the incubation, the constructs are
submerged in epidermalization medium. After a sufficient length of
time in culture, the keratinocytes proliferate and spread to form a
confluent monolayer across the cell-matrix construct. Once
confluent, the cell media formulation is changed to differentiation
medium to induce cell differentiation. When a multilayer epithelium
has formed, cornification media is then used and the culture is
brought to the air-liquid interface. For the differentiation and
cornification of keratinocytes, the cells are exposed to a dry or
low humidity air-liquid interface. A dry or low-humidity interface
can be characterized as trying to duplicate the low moisture levels
of skin. With time, keratinocytes will express most or all keratins
and other features found in native skin when exposed to these
conditions.
[0067] As mentioned above, the system for the production of a
cell-matrix construct may be used in the formation of a corneal
construct. The corneal epithelial cells can be derived from a
variety of mammalian sources. The preferred epithelial cell is a
rabbit or human corneal epithelial cell (corneal keratinocyte) but
any mammalian corneal keratinocyte may be used. Other epithelial
keratinocytes such as those derived from the sclera (outer white
opaque portion) of the eye or epidermis may be substituted, but
corneal keratinocytes are preferable. In the method for forming a
corneal construct, the medium is removed from the culture insert
(containing the cell-matrix construct) and its surround. Normal
rabbit corneal epithelial cells are expanded via subculture,
trypsinized to remove them from the cultures substrate, suspended
in culture medium, and seeded on top of the membrane at a density
between about 7.2.times.10.sup.4 to about 1.4.times.10.sup.5
cells/cm.sup.2. The constructs are then incubated without medium
for about four hours at 37.+-.1.degree. C., 10% CO.sub.2 to allow
the epithelial cells to attach. After incubation, the constructs
are submerged in Corneal Maintenance Medium (CMM) (Johnson et al.,
1992.) The epithelial cells are cultured until the cell-matrix
construct is covered with the epithelial cells. Completeness of
epithelial coverage can be ascertained by a variety of methods, for
illustration by staining the culture with a solution of Nile Blue
sulfate (1:10,000 in phosphate buffered saline). Once the
cell-matrix construct is covered, after approximately seven days,
the constructs are aseptically transferred to new culturing trays
with sufficient cornea maintenance medium (CMM) to achieve a fluid
level just to the surface of the construct to maintain a moist
interface without submersion of the epithelial layer. The
constructs are incubated at 37-1.degree. C., 10% CO.sub.2, and
greater than 60% humidity, with the CMM, making media changes, as
necessary, typically, three times per week.
[0068] For the differentiation, but not the cornification of the
epithelial cell layer, as necessary in the production of a corneal
construct, the epithelial cell surface is exposed to a moist
air-liquid interface. Methods for providing a moist air-liquid
interface are described in U.S. Pat. No. 5,374,515 to Parenteau. As
used herein, the term "moist interface" is intended to mean a
culture environment which is regulated so that the surface of the
construct is moist, with high humidity, but not dry or submerged.
The exact level of moisture and humidity in the culture environment
is not critical, but it should be sufficiently moist and humid to
avoid the formation of cornified cells. A moist interface can be
characterized as trying to duplicate similar moisture levels of the
human eye.
[0069] In an alternate preferred embodiment, a seeding of a second
matrix-producing cell may be performed on a first formed
cell-matrix construct to obtain a thicker cell-matrix construct or
a bilayer cell-matrix construct. The second seeding can be
performed with the same cell type or strain or with a different
cell type or strain, depending on the desired result. The second
seeding is performed under the same conditions employing the
procedures and matrix production medium used in the production of
the first layer. One result in performing the second seeding with a
different cell type is to have a matrix formed with different
matrix component profiles or matrix packing density to affect wound
healing when the construct is grafted to a patient. The first cell
seeding produces a matrix analogous to the reticular layer of
dermis, a more densely packed layer of Type I collagen and
constituent extracellular matrix components. The second cell
seeding would produces a matrix similar to the papillary layer of
dermis characterized by looser collagen fibrils and extracellular
matrix. Another result is the second cell type may produce a
therapeutic substance that would also affect wound healing, such as
improved graft take or graft integration or the summation or
prevention of scar formation.
[0070] In another preferred embodiment, mixed cell populations of
two or more cell types may be cultured together during the
formation of a cell-matrix construct provided that at last one of
the cell types used is capable of synthesizing extracellular
matrix. The second cell type may be one needed to perform other
tissue functions or to develop particular structural features of
the tissue construct. For instance, in the production of a skin
construct, dermal papilla cells or epithelial cells from adnexas
may be cultured with the matrix-producing cells to allow the
formation of epithelial appendages or their components. Epidermal
appendages such as sweat or sebaceous gland structures or
components or hair follicle structures or components may form when
cultured together with the matrix-producing cells. Epithelial cells
may be derived from the appendageal structures of gland and hair
located in deep dermis, such as by microdissection, and include
eccrine cells, myoepithelial cells, glandular secretory cells, hair
follicle stem cells. Other cell types normally found in skin that
constitute skin may also be added such as melanocytes, Langerhans
cells, and Merkel cells. Similarly, vascular endothelial cells may
be co-cultured to produce rudimentary components for new
vasculature formation. Adipocytes may also be cultured with the
matrix-producing cells to form a construct used for reconstructive
surgery. As alternate mode of delivery of this second cell type,
the cells may locally seeded as a spot or as an arrangement of any
number of spots of cells on or within a forming or completely
formed cell-tissue matrix for localized development of these
structures. To seed the cells within the cell matrix construct, the
cells may be injected between the top and bottom surfaces, within
the cell-matrix, for the cells to grow, form specialized structures
and perform their specialized function
[0071] To produce a three-layered tissue construct, a first seeding
of cells comprising a matrix-producing cell type or a
non-matrix-producing cell type is seeded on the culture substrate
for a time sufficient to produce a cell-matrix construct or a cell
layer. Once the first cell-matrix construct or cell layer is
formed, a second seeding of cells comprising a matrix-producing
cell type is seeded on the top surface of the first cell-matrix
construct or cell layer and cultured for a time under conditions
sufficient to form a second cell-matrix construct on the first
construct. On the second cell-matrix construct, a third seeding of
a third cell type is seeded and cultured under sufficient
conditions to produce the third layer. As an example, to produce a
three-layer corneal construct, the cell of the first cell-type may
be comprised of endothelial origin, such as corneal endothelial
cells; the second cell type may comprise cells of connective tissue
origin, such as corneal keratocytes; and the third cell type may
comprise cells of epithelial origin, such as corneal epithelial
cells. As another example of a three-layer construct of skin, the
cell of the first seeding may be of vascular origin to provide
components for vascularization, the cells of the second seeding may
comprise dermal fibroblasts to form a cell-matrix construct to
serve as a dermal construct, and the cells of the third seeding may
be epidermal keratinocytes to form an epidermal layer.
[0072] Tissue constructs of the invention can be stored at
cryogenic temperatures when vitrification or cryopreservation
methods are employed. Methods for vitrification of tissue
constructs are described in U.S. Pat. No. 5,518,878 and methods for
cryopreservation are described in U.S. Pat. Nos. 5,689,961 and
5,891,617 and in International PCT Application WO 96/24018, the
disclosures of which are incorporated herein by reference.
[0073] The skin constructs of this invention can be used in tissue
test systems for in vitro toxicology tests. Test systems that
incorporate skin constructs for testing purposes are described in
U.S. Pat. No. 4,835,102 the disclosure of which is incorporated
herein by reference. Because the cell produced skin construct has
similar structure, and, more importantly, a similar organization to
skin, it can be a valuable test system as an alternative or
replacement to live human or animal testing for absorption,
toxicity, and in many cases effectiveness of products. The
production of the matrix has been shown to mimic several of the
processes exhibited in production of matrix as well as repair of
matrix in vivo. Because of this, the system described can be a
valuable tool in the analysis of wound repair and tissue generation
and further for the testing and analysis of chemical and/or
physical stimulants of wound repair.
[0074] The most preferred use for the skin constructs of this
invention is for grafting or implantation in a mammalian host to
restore or repair the skin due to injury or disease. Indications
for grafting of a skin construct include but are not limited to
plastic or reconstructive surgery, skin wounds, burns, psoriasis,
venous and diabetic ulcers, and basal cell carcinoma. Skin
constructs of the invention are useful to both protect the wounded
tissue, and then serve as a scaffold for the ingrowth of the host
tissue. It is believed that the level of organization of the tissue
produced in this invention would also serve to ease and possibly
speed up the actions of wound repair.
[0075] The cell matrix constructs of the invention have cohesive
properties. "Cohesive" as used herein, means being able to maintain
physical unitary integrity and tissue-like handing properties. The
physical properties that primarily give the constructs of the
invention cohesive properties are bulk thickness and fibrous matrix
structure. The fibrous extracellular matrix is formed from
cell-synthesized collagen and other matrix components, mainly
fibrillar collagen arranged in fibrils and fibril bundles, and
gives the constructs their bulk. The cell-matrix constructs of the
invention are handleable, that is, they can be manually peeled from
their culture substrate, without a carrier support or specialized
tools, and applied to the patient or to a testing apparatus. It can
withstand damage such as tearing or stretching from ordinary
manipulation in the clinic without detriment to the structure or
function. When applied to a patient, they can be secured in place
by sutures or staples.
[0076] To graft the skin construct of the present invention to a
patient, the graft area is prepared according to standard practice.
For burn indications, the burned wound sites to be grafted are to
be prepared for the graft so that the burned skin area is
completely excised. Excised beds will appear clean and clinically
uninfected prior to grafting. For deep partial thickness wounds due
to surgical excision, the preoperative area is shaved, if
necessary, cleansed with an antimicrobial, antiseptic skin cleanser
and rinsed with normal saline. Local anesthesia usually consists of
intradermal administration of lidocaine or epinephrine or both.
Once anesthesia is accomplished, a dermatome is used to remove skin
to an appropriate depth, creating a deep partial thickness wound.
Hemostasis can be achieved by compression with epinephrine
containing lidocaine and by electrocautery. The skin construct is
then applied to the wound bed and, if necessary, is secured by
suturing or stapling in place, then bolstered and bandaged with
appropriate dressings.
[0077] The skin construct of the present invention may also be
meshed prior grafting to a patient. Meshing improves conformation
of the skin construct to the wound bed and provides a means for
draining wound exudate from beneath the graft. The term "meshing"
is defined as a mechanical method by which a tissue is perforated
with slits to form a net-like arrangement. Meshing is preferably
obtained by the use of a conventional skin mesher (ZIMMER.RTM.;
BIOPLASTY.RTM.). One could also manually score or perforate a
tissue with a scalpel or a needle. Meshed skin may be expanded by
stretching the skin so that the slits are opened and then applied
to the wound bed. Expanded meshed tissue provides a wound area with
maximal coverage. Alternatively, meshed skin may be applied without
expansion, simply as a sheet with an arrangement of unexpanded
slits. The meshed skin construct may be applied alone or with the
patient's own skin from another area of the body. Tissue constructs
may also have perforations or fenestrations and pores provided by
other means. Fenestrations may be applied manually using a laser,
punch, scalpel, needle or pin.
[0078] The skin construct of the invention may be applied to wounds
other than surgical wounds or burn areas. Other wounds such as
venous ulcers, diabetic ulcers, decubitus ulcers may experience a
healing benefit by application of the disclosed skin construct.
Other congenital skin diseases such as epidermolysis bullosa may
benefit as well.
[0079] The following examples are provided to better explain the
practice of the present invention and should not be interpreted in
any way to limit the scope of the present invention. Those skilled
in the art will recognize that various modifications can be made to
the methods described herein while not departing from the spirit
and scope of the present invention.
EXAMPLES
Example 1
Formation of a Collagenous Matrix by Human Neonatal Foreskin
Fibroblasts
[0080] Human neonatal foreskin fibroblasts (originated at
Organogenesis, Inc. Canton, Mass.) were seeded at 5.times.10.sup.5
cells/162 cm.sup.2 tissue culture treated flask (Costar Corp.,
Cambridge, Mass., cat #3150) and grown in growth medium. The growth
medium consisted of Dulbecco's Modified Eagle's medium (DMEM) (high
glucose formulation, without L-glutamine, BioWhittaker,
Walkersville, Md.) supplemented with 10% newborn calf serum (NBCS)
(HyClone Laboratories, Inc., Logan, Utah) and 4 mM L-glutamine
(BioWhittaker, Walkersville, Md.). The cells were maintained in an
incubator at 37.+-.1.degree. C. with an atmosphere of 10.+-.1%
CO.sub.2. The medium was replaced with freshly prepared medium
every two to three days. After 8 days in culture, the cells had
grown to confluence, that is, the cells had formed a packed
monolayer along the bottom of the tissue culture flask, and the
medium was aspirated from the culture flask. To rinse the
monolayer, sterile-filtered phosphate buffered saline was added to
the bottom of each culture flask and then aspirated from the
flasks. Cells were released from the flask by adding 5 mL
trypsin-versene glutamine (BioWhittaker, Walkersville, Md.) to each
flask and gently rocking to ensure complete coverage of the
monolayer. Cultures were returned to the incubator. As soon as the
cells were released 5 ml of SBTI (Soybean Trypsin Inhibitor) was
added to each flask and mixed with the suspension to stop the
action of the trypsin-versene. The cell suspension was removed from
the flasks and evenly divided between sterile, conical centrifuge
tubes. Cells were collected by centrifugation at approximately
800-1000.times.g for 5 minutes.
[0081] Cells were resuspended using fresh medium to a concentration
of 3.0.times.10.sup.6 cells/ml, and seeded onto 0.4 micron pore
size, 24 mm diameter tissue culture treated inserts
(TRANSWELL.RTM., Corning Costar) in a six-well tray at a density of
3.0.times.10.sup.6 cells/insert (6.6.times.10.sup.5
cells/cm.sup.2). The cells were maintained in an incubator at
37.+-.1.degree. C. with an atmosphere of 10.+-.1% CO.sub.2 and fed
fresh production medium every 2 to 3 days for 21 days. The
production medium comprised: a 3:1 base mixture of DMEM and Hams
F-12 medium (Quality Biologics Gaithersburg, Md.), 4 mM
GlutaMAX-1.TM. (Gibco BRL, Grand Island, N.Y.) and additives to a
resultant concentration of: 5 ng/ml human recombinant epidermal
growth factor (Upstate Biotechnology Lake Placid, N.Y.), 2% newborn
calf serum (Hyclone, Logan, Utah), 0.4 .mu.g/ml hydrocortisone
(Sigma St. Louis, Mo.), 1.times.10.sup.-4 M ethanolamine (Fluka,
Ronkonkoma, N.Y. ACS grade), 1.times.10.sup.-4 M
o-phosphoryl-ethanolamine (Sigma, St. Louis,), 5 .mu.g/ml insulin
(Sigma, St. Louis, Mo.), 5 .mu.g/ml transferrin (Sigma, St. Louis,
Mo.), 20 .rho.M triiodothyronine (Sigma, St. Louis, Mo.), and 6.78
ng/ml selenium (Sigma Aldrich Fine Chemicals Co., Milwaukee, Wis.),
50 ng/ml L-ascorbic acid (WAKO Chemicals USA, Inc. #013-12061), 0.2
.mu.g/ml. L-proline (Sigma, St. Louis, Mo.), 0.1 .mu.g/ml glycine
(Sigma, St. Louis, Mo.) and 0.05% polyethylene glycol (PEG)
3400-3700 MW (cell culture grade) (Sigma, St. Louis, Mo.).
[0082] Samples for histological analysis were taken at days 7, 14
and 21 and fixed in formalin, then embedded in paraffin. The
formalin fixed samples were embedded in paraffin and 5 micrometer
section were stained with hematoxylin-eosin (H&E) according to
procedures known in the art. Using H&E stained slides,
thickness measurements were made to ten randomly picked microscopic
fields utilizing a 10.times. eyepiece loaded with a 10 mm/100
micrometer reticle.
[0083] Results for two different cell strains of human dermal
fibroblasts are summarized in Table 1, which shows the thickness of
the cell-matrix construct as it develops. TABLE-US-00001 TABLE 1
Thickness (microns) Day 0 Day 7 Day 14 Day 21 B119 0 30.33 .+-.
2.61 63.33 .+-. 4.40 84.00 .+-. 4.67 Average (n = 3) B156 0 42.00
.+-. 5.14 63.85 .+-. 4.50 76.25 .+-. 8.84 Average (n = 4)
[0084] Samples were also submitted for collagen concentration
analysis on days 7, 14, and 21. Collagen content was estimated by
employing a calorimetric assay for hydroxyproline content known in
the art (Woessner, 1961). At those same timepoints cell number was
also determined. Table 2 is a summary of collagen concentration and
Table 3 is a summary of the cell data from cell-matrix constructs
produced from two different cell strains (B156 and B119) using the
procedure described above. TABLE-US-00002 TABLE 2 Collagen
(.mu.g/cm.sup.2) Day 0 Day 7 Day 14 Day 21 B119 0 93.69 .+-. 22.73
241.66 .+-. 21.08 396.30 .+-. 29.38 Average (n = 3) B156 0 107.14
.+-. 17.16 301.93 .+-. 23.91 457.51 .+-. 25.00 Average (n = 3)
[0085] TABLE-US-00003 TABLE 3 Cells (cells/cm.sup.2) Day 0 Day 7
Day 14 Day 21 B119 6.6 .times. 10.sup.5 11.8 .+-. 4.4 .times.
10.sup.5 11.4 .+-. 1.7 .times. 10.sup.5 13.9 .+-. Average 1.2
.times. 10.sup.5 (n = 3) B156 6.6 .times. 10.sup.5 13.1 .+-. 0.5
.times. 10.sup.5 14.0 .+-. 2.1 .times. 10.sup.5 17.1 .+-. Average
1.7 .times. 10.sup.5 (n = 3)
[0086] Samples of the human cell derived dermal matrix from days 7,
14, and 21 were analyzed by delayed reduction SDS-PAGE to determine
collagen composition revealing type I and type III collagen alpha
bands in the samples.
[0087] Biochemical characteristics of the dermal matrix were
determined using immunohistochemical methods. Fibronectin
identification was carried out on paraffin fixed sections using the
Zymed Histostain strepavidin-biotin system (Zymed Laboratories
Inc., South San Francisco, Calif.). Tenascin presence was
determined by primary anti-tenascin antibody staining (Dako,
Carpintheria, Calif.) followed by anti-mouse horseradish peroxidase
labeled antibody (Calbiochem) as a secondary antibody. Samples were
visualized by applying diaminobenzyne (Sigma St. Louis, Mo.) and
counterstained with Nuclear Fast red.
[0088] Glycosaminoglycan (GAG) quantification was performed on 21
day samples using the previously described method (Farndale, 1986).
The assay showed the presence of 0.44 grams of GAG per ca in a
sample of human cell derived dermal matrix taken 21 days post
seeding.
Example 2
Full Thickness Skin Construct
[0089] Using a dermal construct formed using the method described
in Example 1, normal human neonatal foreskin epidermal
keratinocytes (originated at Organogenesis, Inc. Canton, Mass.)
were plated onto the cell-matrix construct to form the epidermal
layer of the skin construct. The medium was aseptically removed
from the culture insert and its surrounds. Normal human epidermal
keratinocytes were scaled up to passage 4 from frozen subculture
cell stock to confluence. Cells were then released from the culture
dishes using trypsin-versene, pooled, centrifuged to form a cell
pellet, resuspended in epidermalization medium, counted and seeded
on top of the membrane at a density of 4.5.times.10.sup.4
cells/cm.sup.2. The constructs are then incubated for 90 minutes at
37.+-.1.degree. C., 10% CO.sub.2 to allow the keratinocytes to
attach. After the incubation, the constructs were submerged in
epidermalization medium The epidermalization medium is composed of
a 3:1 base mixture of Dulbecco's Modified Eagle's Medium (DMEM)
(high glucose formulation, without L-glutamine (BioWhittaker,
Walkersville, Md.) and Hams F-12 medium (Quality Biologics
Gaithersburg, Md.), supplemented with 0.4 .mu.g/ml hydrocortisone
(Sigma St. Louis, Mo.), 1.times.10.sup.-4 M ethanolamine (Fluka,
Ronkonkoma, N.Y.), 1.times.10.sup.-4 M O-phosphoryl-ethanolamine
(Sigma, St. Louis, Mo.), 5 .mu.g/ml insulin (Sigma, St. Louis,
Mo.), 5 .mu.g/ml transferrin (Sigma, St. Louis, Mo.), 20 .mu.M
triiodothyronine (Sigma, St. Louis, Mo.), 6.78 ng/ml selenium
(Aldrich), 24.4 .mu.g/ml adenine (Sigma Aldrich Fine Chemicals
Company, Milwaukee, Wis.), 4 mM L-glutamine (BioWhittaker,
Walkersville, Md.), 0.3% chelated new born calf serum (Hyclone,
Logan, Utah), 0.628 ng/ml progesterone (Amersham Arlington Heights,
Ill.), 50 .mu.g/ml L-ascorbate sodium salt (Sigma Aldrich Fne
Chemicals Company, Milwaukee, Wis.), 10 ng/ml epidermal growth
factor (Life Technologies Inc., MD), and 50 .mu.g/ml gentamycin
sulfate (Amersham, Arlington Heights, Ill.). The constructs were
cultured in the epidermalization medium for 2 days at
37.+-.1.degree. C., 10% CO.sub.2.
[0090] After 2 days the construct was submerged in media composed
of; 3:1 mixture of Dulbecco's modified Eagle's medium (DMEM) (high
glucose formulation, without L-glutamine, BioWhittaker,
Walkersville, Md.), Hams F-12 medium (Quality Biologics,
Gaithersburg, Md.), supplemented with 0.4 .mu.g/ml hydrocortisone
(Sigma, St. Louis, Mo.), 1.times.10.sup.4 ethanolamine (Fluka,
Ronkonkoma, N.Y.), 1.times.10.sup.-4 o-phosphoryl-ethanolamine
(Sigma, St. Louis, Mo.), 5 .mu.g/ml insulin (Sigma, St. Louis,
Mo.), 5 .mu.g/ml transferrin (Sigma, St. Louis, Mo.), 20 .rho.M
triiodothyronine (Sigma, St. Louis, Mo.), and 6.78 ng/ml selenium
(Sigma Aldrich Fne Chemicals Company, Milwaukee, Wis.), 24.4
.mu.g/ml adenine (Sigma Aldrich Fine Chemicals Company), 4 mM
L-glutamine (BioWhittaker, Walkersville, Md.), 0.3% chelated new
born calf serum (BioWhittaker, Walkersville, Md.), 0.628 ng/ml
progesterone (Amersham, Arlington Heights, Ill.), 50 .mu.g/ml
sodium ascorbate, 265 .mu.g/ml calcium chloride (Mallinckrodt,
Chesterfield, Mo.), and 50 .mu.g/ml gentamycin sulfate (Amersham,
Arlington Heights, Ill.). Again the construct was incubated at
37.+-.1.degree. C., 10% CO.sub.2 for 2 days.
[0091] After the 2 days the carrier containing the construct was
aseptically transferred to new culturing trays with a sufficient
amount cornification media, 9 mL, to achieve a fluid level just to
the surface of the carrier membrane to maintain a dry interface to
allow stratification of the epithelial layer. The constructs were
incubated at 37.+-.1.degree. C., 10% CO.sub.2, and low humidity, in
media with media changes every 2-3 days for 7 days. This medium is
composed of; a 1:1 mixture of Dulbecco's modified Eagle's medium
(DMEM) (high glucose formulation, without L-glutamine BioWhittaker,
Walkersville, Md.), Hams P-12 medium (Quality Biologics,
Gaithersburg, Md.), supplemented with 0.4 .mu.g/ml hydrocortisone
(Sigma, St. Louis, Mo.), 1.times.10.sup.-4 M ethanolamine (Fluka,
Ronkonkoma, N.Y.), 1.times.10.sup.-4 M o-phosphoryl-ethanolamine
(Sigma, St. Louis, Mo.), 5 .mu.g/ml insulin (Sigma, St. Louis,
Mo.), 5 .mu.g/ml transferrin (Sigma, St. Louis, Mo.), 20 .rho.M
triiodothyronine (Sigma, St. Louis, Mo.), 6.78 ng/ml selenium
(Aldrich), 24.4 .mu.g/ml adenine (Sigma Aldrich Fine Chemicals
Company), 4 mM L-glutamine (BioWhittaker, Walkersville, Md.), 2%
new born calf serum (BioWhittaker, Walkersville, Md.), 50 .mu.g/ml
sodium ascorbate, and 50 .mu.g/ml gentamycin sulfate (Amersham,
Arlington Heights, Ill.). After 7 days the construct was fed for 10
more days, with changes every 2-3 days with a maintenance medium.
This maintenance medium was composed of; 1:1 mixture of Dulbecco's
modified Eagle's medium (DMEM) (high glucose formulation, without
L-glutamine, BioWhittaker, Walkersville, Md.), Hams F12 medium
(Quality Biologics Gaithersburg, Md.), 0.4 .mu.g/ml hydrocortisone
(Sigma St. Louis, Mo.), 1.times.10.sup.-4 M ethanolamine (Fluka,
Ronkonkoma, N.Y.), 1.times.10.sup.-4 M o-phosphoryl-ethanolamine
(Sigma, St. Louis, Mo.), 5 .mu.g/ml insulin (Sigma, St. Louis,
Mo.), 5 .mu.g/ml transferin (Sigma, St. Louis, Mo.), 20 .rho.M
triiodothyronine (Sigma, St. Louis, Mo.), and 6.78 ng/ml selenium
(Sigma Aldrich Fine Chemicals Company, Milwaukee, Wis.), 24.4
.mu.g/ml adenine (Sigma Aldrich Fine Chemicals Company, Milwaukee,
Wis.), 4 mM L-glutamine (BioWhittaker, Walkersville, Md.), 1% new
born calf serum (BioWhittaker, Walkersville, Md.), and 50 .mu.g/ml
gentamycin sulfate (Amersham, Arlington Heights, Ill.).
[0092] Final samples were submitted for hemotoxylin and eosin
processing as described in Example 1 to determine gross appearance
under light microscopy. The resulting construct consisted of a
lower (dermal) layer consisting of fibroblasts surrounded by matrix
having features described in Example 1, and was completely overlaid
by a multilayered, stratified and well-differentiated layer of
keratinocytes that exhibit a basal layer, a suprabasal layer, a
granular layer and a stratum corneum similar to that of skin in
situ. The skin construct has a well-developed basement membrane
present at the dermal-epidermal junction as exhibited by
transmission electron microscopy (TEM). The basement membrane
appears thickest around hemidesmosomes, marked by anchoring fibrils
that are comprised of type VII collagen, as visualized by TEM. As
expected these anchoring fibrils can easily be seen exiting from
the basement membrane and entrapping the collagen fibrils. The
presence of laminin, a basement membrane glycoprotein, was shown
using the previously described avidin-biotin immunoenzymatic
technique (Guesdon, 1979).
Example 3
In Vitro Formation of a Collagenous Matrix by Human Neonatal
Foreskin Fibroblasts in Chemically Defined Medium
[0093] Human neonatal foreskin fibroblasts were expanded using the
procedure described in Example 1. Cells were then resuspended to a
concentration of 3.times.10.sup.6 cells/ml, and seeded on to 0.4
micron pore size, 24 mm diameter tissue culture treated membrane
inserts in a six-well tray at a density of 3.0.times.10.sup.6
cells/TW (6.6.times.10.sup.5 cells/cm.sup.2). These cells were then
maintained as Example 1 with newborn calf serum omitted from the
media throughout. More specifically the medium contained: a base
3:1 mixture of DMEM, Hams F12 medium (Quality Biologics,
Gaithersburg, Md.), 4 mM GlutaMAX (Gibco BRL, Grand Island, N.Y.)
and additives: 5 ng/ml human recombinant epidermal growth factor
(Upstate Biotechnology, Lake Placid, N.Y.), 0.4 .mu.g/ml
hydrocortisone (Sigma, St. Louis, Mo.), 1.times.10.sup.-4 M
ethanolamine (Fluka, Ronkonkoma, N.Y. cat. #02400 ACS grade),
1.times.10.sup.-4 M o-phosphoryl-ethanolamine (Sigma, St. Louis,
Mo.), 5 .mu.g/ml insulin (Sigma, St. Louis, Mo.), 5 .mu.g/ml
transferrin (Sigma, St. Louis, Mo.), 20 .rho.M triiodothyronine
(Sigma, St. Louis, Mo.), and 6.78 ng/ml selenium (Sigma Aldrich
Fine Chemicals Company, Milwaukee, Wis.), 50 ng/ml L-ascorbic acid
(WAKO Chemicals USA, Inc.), 0.2 .mu.g/ml L-proline (Sigma, St.
Louis, Mo.), 0.1 .mu.g/ml glycine (Sigma, St. Louis, Mo.) and 0.05%
poly-ethylene glycol (PEG) (Sigma, St. Louis, Mo.). Samples were
checked at day 7, 14, and 21 for collagen concentration and cell
number using described procedures. Results are summarized in tables
4 (cell number) and 5 (collagen). Samples were also formalin fixed
and processed for hemotoxylin and eosin staining for light
microscope analysis as described in Example 1. Histological
evaluation demonstrated that the constructs grown in defined medium
was similar to those grown in the presence of 2% newborn calf
serum. Samples also stained positively for fibronectin, using
procedure described in Example 1. TABLE-US-00004 TABLE 4 Collagen
(.mu.g/cm.sup.2) Day 0 Day 7 Day 14 Day 21 Average 0 107.63 .+-.
21.96 329.85 .+-. 27.63 465.83 .+-. 49.46 amount of collagen in
each construct (n = 3)
[0094] TABLE-US-00005 TABLE 5 Cells (cells/cm.sup.2) Day 0 Day 7
Day 14 Day 21 Average 6.6 .times. 10.sup.5 7.8 .+-. 2.2 .times.
10.sup.5 9.6 .+-. 2.5 .times. 10.sup.5 1.19 .+-. number of 2.1
.times. 10.sup.5 cells in each construct (n = 3)
[0095] Besides endogenously produced fibrillar collagen, decorin
and glycosaminoglycan were also present in the cell-matrix
construct.
Example 4
Full Thickness Skin Construct Formed Using Chemically Defined
Media
[0096] Using a 25 day dermal construct formed by human dermal
fibroblasts under chemically defined conditions similar to the
method described in Example 3, normal human neonatal foreskin
epidermal keratinocytes were seeded on the top surface of the
cell-matrix construct to form the epidermal layer of the skin
construct.
[0097] The medium was aseptically removed from the culture insert
and its surrounds. Normal human epidermal keratinocytes were scaled
up to passage 4 from frozen subculture cell stock to confluence.
Cells were then released from the culture dishes using
trypsin-versene, pooled, centrifuged to form a cell pellet,
resuspended in epidermalization medium, counted and seeded on top
of the membrane at a density of 4.5.times.10.sup.4 cells/cm.sup.2.
The constructs were then incubated for 90 minutes at
37.+-.1.degree. C., 10% CO.sub.2 to allow the keratinocytes to
attach. After the incubation, the constructs were submerged in
epidermalization medium. The epidermalization medium is composed of
a 3:1 base mixture of Dulbecco's Modified Eagle's Medium (DMEM)
(containing no glucose and no calcium, BioWhittaker, Walkersville,
Md.) and Hams F-12 medium (Quality Biologics Gaithersburg, Md.),
supplemented with 0.4 .mu.g/ml hydrocortisone (Sigma St. Louis,
Mo.), 1.times.10.sup.-4 M ethanolamine (Fluka, Ronkonkoma, N.Y.),
1.times.10.sup.-4 M o-phosphoryl-ethanolamine (Sigma, St. Louis,
Mo.), 5 .mu.g/ml insulin (Sigma, St. Louis, Mo.), 5 .mu.g/ml
transferrin (Sigma, St. Louis, Mo.), 20 .rho.M triiodothyronine
(Sigma, St. Louis, Mo.), 6.78 ng/ml selenium (Aldrich), 24.4
.mu.g/ml adenine (Sigma Aldrich Fine Chemicals Company, Milwaukee,
Wis.), 4 mM L-glutamine (BioWhittaker, Walkersville, Md.), 50
.mu.g/ml L-ascorbate sodium salt (Sigma Aldrich Fine Chemicals
Company, Milwaukee, Wis.), 16 .mu.M linoleic acid (Sigma, St.
Louis, Mo.), 1 .mu.M tocopherol Acetate (Sigma, St. Louis, Mo.) and
50 .mu.g/ml gentamicin sulfate (Amersham, Arlington Heights, Ill.).
The constructs were cultured in the epidermalization medium for 2
days at 37.+-.1.degree. C., 10.+-.1% CO.sub.2.
[0098] After 2 days the medium was exchanged with fresh medium
composed as above, and returned to the incubator set at
37.+-.1.degree. C., 10.+-.1% CO.sub.2 for 2 days. After the 2 days,
the carrier containing the construct was aseptically transferred to
new culturing trays with sufficient media to achieve a fluid level
just to the surface of the carrier membrane to maintain the
developing construct at the air-liquid interface. The air
contacting the top surface of the forming epidermal layer allows
stratification of the epithelial layer. The constructs were
incubated at 37.+-.1.degree. C., 10% CO.sub.2, and low humidity, in
media with media changes every 2-3 days for 7 days. This medium
contained a 1:1 mixture of Dulbecco's modified Eagle's medium
(DMEM) (containing no glucose and no calcium, BioWhittaker,
Walkersville, Md.), Hams F-12 medium (Quality Biologics,
Gaithersburg, Md.), supplemented with 0.4 .mu.g/ml hydrocortisone
(Sigma, St. Louis, Mo.), 5.times.10.sup.-4 M ethanolamine (Fluka,
Ronkonkoma, N.Y.), 5.times.10.sup.-4 M o-phosphoryl-ethanolamine
(Sigma, St. Louis, Mo.), 5 .mu.g/ml insulin (Sigma, St. Louis,
Mo.), 5 .mu.g/ml transferrin (Sigma, St. Louis, Mo.), 20 .rho.M
triiodothyronine (Sigma, St. Louis, Mo.), 6.78 ng/ml selenium
(Sigma Aldrich Fine Chemicals Company), 24.4 .mu.g/ml adenine
(Sigma Aldrich Fine Chemicals Company), 4 mM L-glutamine
(BioWhittaker, Walkersville, Md.), 2.65 .mu.g/ml calcium chloride
(Mallinckrodt, Chesterfield, Mo.), 16 .mu.M linoleic acid (Sigma,
St. Louis, Mo.), 1 .mu.M tocopherol acetate (Sigma, St. Louis,
Mo.), 1.25 mM serine (Sigma, St. Louis, Mo.), 0.64 mM choline
chloride (Sigma, St. Louis, Mo.) and 50 .mu.g/ml gentamicin sulfate
(Amersham, Arlington Heights, Ill.). The cultures were fed every
2-3 days, for 14 days.
[0099] Samples, in triplicate, were submitted 10, 12, and 14 days
after the construct was lifted to the air-liquid interface for
hematoxylin and eosin processing as described in Example 1 to
determine gross appearance under light microscopy. The resulting
construct consisted of a lower (dermal) layer consisting of
fibroblasts surrounded by matrix having features as described in
Example 3, and was overlaid by a layer of stratified and
differentiated keratinocytes.
Example 5
In Vitro Formation of a Collagenous Matrix by Human Achilles Tendon
Fibroblasts
[0100] Cell-matrix constructs were formed using the same method
described in Example 1 replacing the human neonatal foreskin
fibroblasts with human Achilles tendon fibroblasts (HATF.).
Following 21 days in production medium, samples were also submitted
for H&B staining and thickness determination using the
procedure described in Example 1. The resulting construct was
visualized as a cell matrix tissue like construct with a thickness
of 75.00.+-.27.58 microns (n=2). Endogenously produced fibrillar
collagen, decorin and glycosaminoglycan were also present in the
construct.
Example 6
In Vitro Formation of a Collagenous Matrix by Transfected Human
Neonatal Foreskin Fibroblasts
[0101] Transfected human dermal fibroblasts were produced using the
following procedure. One vial of jCRIP-43 platelet derived growth
factor (PDGF) viral producers (Morgan, J, et al.) was thawed, and
the cells were seeded at 2.times.10.sup.6 cells/162 cm.sup.2 flask
(Corning Costar, Cambridge, Mass.). These flasks were fed a growth
medium, and maintained in an incubator at 37.+-.1.degree. C. with
an atmosphere of 10.+-.1% CO.sub.2. The growth medium consisted of
Dulbecco's modified Eagle's medium (DMEM) (high glucose
formulation, without L-glutamine, BioWhittaker, Walkersville, Md.)
supplemented with 10% newborn calf serum (HyClone Laboratories,
Inc., Logan, Utah) and 4 mM L-glutamine (BioWhittaker,
Walkersville, Md.). On the same day, 1 vial of human neonatal
foreskin fibroblast (HDFB156) was also thawed and plated at
1.5.times.10.sup.6 cells/162 cm.sup.2 flask (Corning Costar,
Cambridge, Mass.). After three days the jCRIP PDGF-43 viral
producers were fed with fresh growth medium. The HDFB156 were fed
with the above growth medium plus 8 .mu.g/ml polybrene (Sigma, St.
Louis, Mo.). The next day the HDFB156's cells were infected as
follows. The spent medium from the jCRIP PDGF-43 viral producers
was collected and filtered through a 0.45 micron filter. 8 .mu.g/ml
polybrene was added to this filtered spent medium. The spent medium
was then placed on the HDF. On the next two days the HDF were fed
fresh growth medium. The day after the HDF were passed from p5 to
p6 and seeded at a density of 2.5.times.10.sup.6 cells/162 cm.sup.2
flask (Corning Costar, Cambridge, Mass.). Cells were passed as
follows; spent medium was aspirated off. The flasks were then
rinsed with a phosphate buffered saline to remove any residual
newborn calf serum. Cells were released from the flask by adding 5
mL trypsin-versene to each flask and gently rocking to ensure
complete coverage of the monolayer. Cultures were returned to the
incubator. As soon as the cells were released, 5 mL of SBTI
(Soybean Trypsin Inhibitor) was added to each flask and mixed with
the suspension to stop the action of the trypsin-versene. The
cell/Trypsin/SBTI suspension was removed from the flasks and evenly
divided between sterile, conical centrifuge tubes. Cells were
collected by centrifugation at approximately 800-1000.times.g for 5
minutes.). The cells were resuspended in the growth media for
seeding at the density listed above. After two days the cells were
fed fresh growth medium. The following day the cells were harvested
as above, and diluted to a density of 1.5.times.10.sup.6 cells/ml
in growth medium containing 10% newborn calf serum (NBCS) with 10%
dimethyl sulfoxide (DMSO) (Sigma, St. Louis, Mo.). The cells were
then frozen 1 m/cryovial at about 40.degree. C. Production of the
collagenous matrix for this example utilize the same procedure as
Examples 1 and 3, replacing the human neonatal foreskin fibroblasts
with human neonatal foreskin fibroblasts transformed to produce
high levels of platelet derived growth factor (PDGF) as described
above. Samples were taken for H&E staining as described above
on day 18 post seeding. Samples were also stained using the
avidin-biotin methods for the presence of fibronectin listed in
Example 10. Samples were taken on day 18 post seeding for H&E
staining as described in Example 1, and exhibited a similar
cell-matrix gross appearance to that described in Example 1, with a
measured thickness of 123.6 microns (N=1). PDGF output of the
transfected cells in the cell-matrix construct was measured to be
100 ng/mL by ELISA throughout the duration of the culture (18 days)
while control output of PDGF was undetectable.
Example 7
Use of The Dermal Construct as a Graft Material
[0102] Cell-matrix constructs were prepared according to the
methods in Example 1 using human dermal fibroblasts derived from
neonate foreskin and were grafted onto full excision wounds created
on nude athymic mice. Mice were grafted according to the methods
described by Parenteau, et al. (1996), the disclosure of which is
incorporated herein. Grafts were examined at 14, 28 and 56 days for
signs of adherence to the wound bed, evidence of wound contraction,
areas of graft loss, and presence of vascularization (color). The
graft areas were photographed while intact on the mice. A number of
mice were sacrificed at each timepoint, and the graft areas and
their surrounds were excised along with a surrounding rim of murine
skin to at least the panniculus carnosus. Junctions between the
graft and the murine skin were preserved in each sample. The
explanted tissue samples were then fixed in phosphate buffered 10%
formalin and fixation in methanol. Formal fixed samples were
processed for H&E staining according to procedure described in
Example 1. Grafts were able to integrate with the mouse skin, with
minimal contraction noted. Within 14 days of grafting, the mouse
epidermis had migrated completely over the graft. Using the H&B
stained samples, vessels were obvious within the graft at 14 days,
and throughout the experiment. By gross observation and by H&E
stained samples, it was determined that the graft persisted and
remained healthy looking contained living cells, no gross matrix
abnormalities, etc.) throughout the length of the experiment.
Example 8
Use of Full Thickness Skin Construct as a Skin Graft
[0103] Bilayer skin constructs were prepared as described in
Example 2 using human dermal fibroblasts derived from neonate
foreskin in the dermal layer and human keratinocytes derived from a
different neonate foreskin in the epidermal layer. The skin
constructs were able to be manually peeled from the membrane,
handled without carrier support, and placed onto the graft site.
The bilayer skin constructs were grafted onto full excision wounds
created on athymic nude mice according to the methods described by
Parenteau, et al. (1996), the disclosure of which is incorporated
herein. Timepoints for taking samples were days 7, 14, 28, 56, and
184 days post-graft. The graft areas were photographed while intact
on the mice. A number of mice were sacrificed at each timepoint,
and the graft areas and their surrounds were excised along with a
surrounding rim of murine skin to at least the panniculus carnosus.
Junctions between the graft and the murine skin were preserved in
each sample. The explanted tissue samples were then fixed in
phosphate buffered 10% formalin and fixation in methanol. Formalin
fixed samples were processed for H&E staining according to
procedure described in Example 1.
[0104] The grafts integrated with the host tissue within 7 days by
gross observation as well as by histological appearance. By H&E
staining, vessels were visualized growing into the graft from the
host tissue within 7 days of grafting. The grafts remained healthy
and persisted through the experiment, with minimal contraction
noted. Utilizing anti-human Involucrin staining the persistence of
human epidermal cells was shown for the entire graft period.
Example 9
In Vitro Formation of a Matrix by Human Corneal Keratocytes
[0105] Human corneal keratocyte cells (originated at Organogenesis,
Inc. Cantor, Mass.) were used in the production of a stromal
construct of cornea. Confluent cultures of human keratocytes were
released from their culture substrates using trypsin-versene. When
released, soybean trypsin inhibitor was used to neutralize the
trypsin-versene, the cell suspension was centrifuged, the
supernatant discarded and the cells were then resuspended in base
media to a concentration of 3.times.10.sup.6 cells/ml. Cells were
seeded onto 0.4 micron pore size, 24 mm diameter tissue culture
treated transwells in a six-well tray at a density of
3.0.times.10.sup.6 cells/TW (6.6.times.10.sup.5 cells/cm.sup.2).
These cultures were maintained overnight in seed medium. The seed
medium was composed of a base 3:1 mixture of Dulbecco's Modified
Eagle's Medium (DMEM) and Hams 12 Medium (Quality Biologics
Gaithersburg, Md. cat.), 4 mM GlutaMAX (Gibco BRL, Grand Island,
N.Y.) and additives: 5 ng/ml human recombinant epidermal growth
factor (EGF) (Upstate Biotechnology Lake Placid, N.Y.), 0.4
.mu.g/ml hydrocortisone (Sigma St. Louis, Mo.), 1.times.10.sup.-4 M
ethanolamine (Fluka, Ronkonkoma, N.Y.), 1.times.10.sup.-4 M
o-phosphoryl-ethanolamine (Sigma, St. Louis, Mo.), 5 .mu.g/ml
insulin (Sigma, St. Louis, Mo.), 5 .mu.g/ml transferrin (Sigma, St.
Louis, Mo.), 20 .rho.M triiodothyronine (Sigma, St. Louis, Mo.),
and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Company,
Milwaukee, Wis.). Following this the cultures were fed flesh
production medium. The production medium was composed of: a base
3:1 mixture of DMEM, Hams F-12 medium (Quality Biologics
Gaithersburg, Md.), 4 mM GlutaMAX (Gibco BRL., Grand Island, N.Y.)
and additives: 5 ng/ml Human Recombinant Epidermal growth factor
(Upstate Biotechnology Lake Placid, N.Y.), 2% newborn calf serum
(Hyclone, Logan, Utah), 0.4 .mu.g/ml hydrocortisone (Sigma, St.
Louis, Mo.), 1.times.10.sup.-4 M ethanolamine (Fluka, Ronkonkoma,
N.Y. ACS grade), 1.times.10.sup.-4 M o-phosphoryl-ethanolamine
(Sigma, St. Louis,), 5 .mu.g/ml insulin (Sigma, St. Louis, Mo.), 5
.mu.g/ml transferrin (Sigma, St. Louis, Mo.), 20 .rho.M
triiodothyronine (Sigma, St. Louis, Mo.), and 6.78 ng/ml selenium
(Sigma Aldrich Fine Chemicals Co., Milwaukee, Wis.), 50 ng/ml
L-ascorbic acid (WAKO pure chemical company), 0.2 .rho.g/ml
L-proline (Sigma, St. Louis, Mo.), 0.1 .mu.g/ml glycine (Sigma, St.
Louis, Mo.) and 0.05% poly-ethylene glycol (PEG) (Sigma, St. Louis,
Mo., cell culture grade).
[0106] The cells were maintained in an incubator at 37.+-.1.degree.
C. with an atmosphere of 10%.+-.1% CO.sub.2 and fed fresh
production medium every 2-3 days for 20 days (for a total of 21
days in culture. After 21 days in culture, the keratocytes had
deposited a matrix layer of about 40 microns in thickness, as
measured by the method described in Example 1. Endogenously
produced fibrillar collagen, decorin and glycosaminoglycan were
also present in the cell-matrix construct.
Example 10
In Vitro Formation of a Collagenous Matrix by Human Neonatal
Foreskin Fibroblasts Seeded in Production Media
[0107] Human neonatal foreskin fibroblasts (originated at
Organogenesis, Inc. Canton, Mass.) were seeded at 1.times.10.sup.5
cells/0.4 micron pore size, 24 mm diameter tissue culture treated
carriers in a six-well tray (TRANSWELL.RTM., Costar Corp.
Cambridge, Mass.) and grown in growth medium. The growth medium
consisted of Dulbecco's Modified Eagle's medium (DMEM) (high
glucose formulation, without L-glutamine, BioWhittaker,
Walkersville, Md.) supplemented with 10% newborn calf serum
(HyClone Laboratories, Inc., Logan, Utah) and 4 mM L-Glutamine
(BioWhittaker, Walkersville, Md.). The cells were maintained in an
incubator at 37.+-.1.degree. C. with an atmosphere of 10.+-.1%
CO.sub.2. The medium was replaced every two to three days. After 9
days in culture the medium was aspirated from the culture dish, and
replaced with production medium. The cells were maintained in an
incubator at 37.+-.1.degree. C. with an atmosphere of 10.+-.1%
CO.sub.2 and fed fresh production medium every 2-3 days for 21
days. The production medium was composed of: a base 3:1 mixture of
DMEM, Hams F-12 medium (Quality Biologics, Gaithersburg, Md.), 4 mM
GlutaMAX (Gibco BRL, Grand Island, N.Y.) and additives: 5 ng/ml
human recombinant epidermal growth factor (Upstate Biotechnology,
Lake Placid, N.Y.), 2% newborn calf serum (Hyclone, Logan, Utah),
0.4 .mu.g/ml hydrocortisone (Sigma St. Louis, Mo.),
1.times.10.sup.-4 M ethanolamine (Fluka, Ronkonkoma, N.Y. ACS
grade), 1.times.10.sup.-4 M o-phosphoryl-ethanolamine (Sigma, St.
Louis,), 5 .mu.g/ml insulin (Sigma, St. Louis, Mo.), 5 .mu.g/ml
transferrin (Sigma, St. Louis, Mo.), 20 .mu.M triiodothyronine
(Sigma, St. Louis, Mo.), and 6.78 ng/ml selenium (Sigma Aldrich
Fine Chemicals Co., Milwaukee, Wis.), 50 ng/ml L-ascorbic acid
(WAKO Pure Chemical Company), 0.2 .mu.g/ml L-proline (Sigma, St.
Louis, Mo.), 0.1 .mu.g/ml glycine (Sigma, St. Louis, Mo.) and 0.05%
polyethylene glycol (PEG) (Sigma, St. Louis, Mo., cell culture
grade).
[0108] Samples were taken at day 21 and fixed in formalin, then
embedded in paraffin. The formalin fixed samples were embedded in
paraffin and 5 micrometer section were stained with
hematoxylin-eosin (H&E) according techniques routinely used in
the art. Using H&E stained slides, measurements were made at
ten randomly picked microscopic fields utilizing a 10.times.
Eyepiece (Olympus America Inc., Melville, N.Y.) loaded with a 10
mm/100 micrometer reticle (Olympus America Inc., Melville, N.Y.).
The constructs created using this method are similar in structure
and biochemical composition to those created with Example 1, and
have a measured thickness of 82.00.+-.7.64 microns.
Example 11
In Vitro Formation of a Collagenous Matrix by Pig Dermal
Fibroblasts
[0109] Pig Dermal Fibroblasts (originated at Organogenesis, Inc.
Canton, Mass.) were seeded at 5.times.10.sup.5 cells/162 cm.sup.2
tissue culture treated flask (Costar Corp., Cambridge, Mass. cat
#3150) and grown in growth medium as described below. The growth
medium consisted of; Dulbecco's modified Eagle's medium (DMEM)
(high glucose formulation, without L-glutamine, BioWhittaker,
Walkersville, Md.) supplemented with 10% fetal calf serum (HyClone
Laboratories, Inc., Logan, Utah) and 4 mM L-glutamine
(BioWhittaker, Walkersville, Md.). The cells were maintained in an
incubator at 37.+-.1.degree. C. with an atmosphere of 10%.+-.1% M.
The medium was replaced every two to three days. Upon confluence,
that is the cells had formed a packed layer at the bottom of the
tissue culture flask, the medium was aspirated from the culture
dish. To rinse the monolayer, sterile-filtered phosphate buffered
saline was added to the monolayer and then aspirated from the dish.
Cells were released from the flask by adding 5 ml trypsin-versene
glutamine (BioWhittaker, Walkersville, Md.) to each flask and
gently rocking to ensure complete coverage of the monolayer.
Cultures were returned to the incubator. As soon as the cells were
released 5 ml of SBTI (Soybean Trypsin Inhibitor) was added to each
flask and mixed with the cell suspension to stop the action of the
trypsin-versene. The suspension was removed from the flasks and
evenly divided between sterile, conical centrifuge tubes. Cells
were collected by centrifugation at approximately 800-1000.times.g
for 5 minutes. Cells were resuspended and diluted to a
concentration of 3.times.10.sup.6 cell/ml, and seeded onto 0.4
micron pore size, 24 mm diameter tissue culture treated transwells
in a six-well tray at a density of 3.0.times.10.sup.6 cells/TW
(6.6.times.10.sup.5 cells/cm.sup.2). Cells were maintained
overnight in a seed medium. The seed medium consisted of, a base
3:1 mixture of DMEM, Hams F-12 medium (Quality Biologics,
Gaithersburg, Md.), 4 mM GlutaMAX (Gibco BRL, Grand Island, N.Y.)
and additives: 5 ng/ml human recombinant epidermal growth factor
(Upstate Biotechnology Lake Placid, N.Y.), 0.4 .mu.g/ml
hydrocortisone (Sigma St. Louis, Mo.), 1.times.10.sup.-4 M
ethanolamine (Fluka, Ronkonkoma, N.Y. ACS grade), 1.times.10.sup.-4
M o-phosphoryl-ethanolamine (Sigma, St. Louis,), 5 .mu.g/ml insulin
(Sigma, St. Louis, Mo.), 5 .mu.g/ml transferrin (Sigma, St. Louis,
Mo.), 20 .rho.M triiodothyronine (Sigma, St. Louis, Mo.), and 6.78
ng/ml selenium (Sigma Aldrich Fine Chemicals Co., Milwaukee, Wis.),
50 ng/ml L-ascorbic acid (WAKO Pure Chemical Company), 0.2 .mu.g/ml
L-proline (Sigma, St. Louis, Mo.), and 0.1 .mu.g/ml glycine (Sigma,
St. Louis, Mo.). The cells were maintained in an incubator at
37.+-.1.degree. C. with an atmosphere of 10.+-.1% CO.sub.2 and fed
fresh production medium every 2-3 days for 7 days. The production
medium was composed of: a base 3:1 mixture of DMEM, Hams F12 medium
(Quality Biologics, Gaithersburg, Md.), 4 mM GlutaMAX (Gibco BRL,
Grand Island, N.Y.) and additives: 5 ng/ml human recombinant
epidermal growth factor (Upstate Biotechnology, Lake Placid, N.Y.),
2% newborn calf serum (Hyclone, Logan, Utah), 0.4 .mu.l/ml
hydrocortisone (Sigma St. Louis, Mo.), 1.times.10.sup.-4 M
ethanolamine (Fluka, Ronkonkoma, N.Y. ACS grade), 1.times.10.sup.-4
M o-phosphoryl-ethanolamine (Sigma, St. Louis,), 5 .mu.g/m insulin
(Sigma, St. Louis, Mo.), 5 .mu.g/ml transferrin (Sigma, St. Louis,
Mo.), 20 .mu.M triiodothyronine (Sigma, St. Louis, Mo.), and 6.78
ng/ml selenium (Sigma Aldrich Fine Chemicals Co., Milwaukee, Wis.),
50 ng/ml L-ascorbic acid (WAKO Pure Chemical Company), 0.2 .mu.g/ml
L-proline (Sigma, St. Louis, Mo.), 0.1 .mu.g/ml glycine (Sigma, St.
Louis, Mo.) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis,
Mo.) cell culture grade. After 7 days the media was replaced with
production medium without newborn calf serum. This media was fed
fresh to the cells every 2-3 days for 20 more days, for a total of
28 days in culture.
[0110] Samples were taken at day 21 and fixed in formalin, then
embedded in paraffin. The formalin fixed samples were embedded in
paraffin and 5 micrometer section were stained with
hematoxylin-eosin (H&E) according to techniques customarily
used in the art. Using H&E stained slides, measurements were
made at ten randomly picked microscopic fields utilizing a
10.times. Eyepiece (Olympus America Inc., Melville, N.Y.) loaded
with a 10 mm 100 micrometer reticle (Olympus America Inc.,
Millville, N.Y.). The sample exhibited a structure composed of
cells and matrix with a measured thickness of 71.20.+-.9.57
microns. Besides endogenously produced fubrillar collagen, decorin
and glycosaminoglycan were also present in the cell-matrix
construct.
Example 12
In Vitro Formation of a Bilayer Skin Construct Containing Cells of
Dermal Papilla
[0111] A cell-matrix was made according to the method in Example 1
using Human Neonatal Foreskin Fibroblasts as a first matrix
producing cell type. The cell-matrix was locally seeded with spots
of dermal papilla cells as a second cell population which was in
turn seeded with keratinocytes as a third cell population, to form
a continuous epidermal layer over the cell-matrix and the dermal
papilla cells.
[0112] First, a cell-matrix construct was formed using human dermal
fibroblasts (HDF) derived from neonatal foreskin. HDF were scaled
up by seeding them at 5.times.10.sup.5 cells/162 cm.sup.2 tissue
culture treated flask (Costar Corp., Cambridge, Mass.) in growth
medium consisting of Dulbeco's Modified Eagle's medium (DMEM) (high
glucose formulation, without L-glutamine, BioWhittaker,
Walkersville, Md.) supplemented with 10% newborn calf serum (NBCS)
(HyClone Laboratories, Inc., Logan, Utah) and 4 mM L-glutamine
(BioWhittaker, Walkersville, Md.). When confluent, HDF were
released from the plate using trypsin-versene and resuspended using
fresh medium to a concentration of 3.0.times.10.sup.6 cells/ml, and
seeded onto 0.4 micron pore size, 24 mm diameter tissue culture
treated inserts TRANSWELL.RTM., Corning Costar) in a six-well tray
at a density of 3.0.times.10.sup.6 cells/insert (6.6.times.10.sup.5
cells/cm.sup.2). HDF cultures were maintained in an incubator at
37.+-.1.degree. C. with an atmosphere of 10.+-.1% CO.sub.2 and fed
fresh production medium every 2 to 3 days for 23 days according the
method detailed in Example 1.
[0113] After the cell-matrix construct had formed, it was seeded
with spots of dermal papillae cells as a second cell population.
Dermal papilla cells are a discrete population of specified
fibroblasts surrounded by the hair bulb of hair follicles to play a
support role in the hair growth. Dermal papillae can be isolated by
microdissecting hair follicles and cultured in vitro using the
method previously described by Messenger, A. G., The Culture of
Dermal Papilla Cells from Human Hair Follicles. Br. J. Dermatol.
110: 685-9 (1984), the method of which is incorporated herein. When
a culture of dermal papilla cells reach confluence they form
aggregates that can be replated on culture flasks to reform new
aggregates. Dermal papillae were isolated from a skin biopsy
obtained from a 4 week old pig. Cells from the dermal papilla (PDP)
were serially cultured in DMEM containing 20% of NBCS until passage
8. After 3 weeks in culture, the PDP cells reformed dermal
papilla-like structures, or aggregates, that each had a diameter
approximately between 90 to 210 microns. The aggregates were then
removed from the culture plate by vigorous pipetting of medium
against them, and then seeded onto the Human Collagenous Matrix at
the density of 200 aggregates per cm.sup.2. The aggregates were
cultured submerged for an additional 15 days in DMEM 20% NBCS with
spent medium exchanged with fresh medium every 2-3 days.
[0114] The cell-matrix cultures containing dermal papilla cells
thereon were seeded with keratinocytes and cultured to form a
continuous epidermal layer over the cell-matrix and the dermal
papillae. Two different constructs were made: the first with human
keratinocytes, the second with pig keratinocytes. Normal epidermal
keratinocytes were isolated from human neonatal foreskin (HEP), or
from pig keratinocytes (PEP) using explant outgrowth to establish
primary cultures. These cells were then cultured and expanded until
passage 3 for the pig strain, or until passage 4 for the human
strain. After about 5 to 6 days in culture, cells were then
released from the culture dishes using trypsin-versene, pooled,
centrifuged to form a cell pellet, resuspended in epidermalization
medium, counted and seeded on top of the membrane at a density of
4.5.times.10.sup.4 cells/cm.sup.2 for HEP cells, or
1.6.times.10.sup.5 cells/cm.sup.2 for HEP cells. Epidermalized
cultures were cultured for 12 days as previously described in
Example 2.
[0115] Final samples were submitted for hematoxylin and eosin
processing for light microscopy. The resulting skin constructs
exhibited the basic morphological organization similar to skin: a
dermal layer consisting of fibroblasts surrounded by endogenously
produced matrix, including endogenously produced fibrillar
collagen, decorin and glycosaminoglycan, localized areas of dermal
papilla cells and a continuous, stratified layer of keratinocytes
across the cell-matrix construct and the dermal papillae. In both
tissue constructs overlaid with either human or pig keratinocytes,
the dermal papilla maintained a packed structure that induced small
undulations of the overlaid epithelium. Differentiated epithelial
cells are often present close to the dermal papilla cells.
Example 13
Hyaluronic Acid Measurement by Sandwich ELISA
[0116] Hyaluronic acid (HA) was measured in cell-matrix constructs
formed by dermal fibroblasts in serum-containing medium and
chemically defined medium according to the methods of Examples 1
and 3, respectively.
[0117] Cell-matrix constructs were formed on circular 75 mm
diameter carriers incorporating a porous membrane (TRANSWELL.RTM.,
Corning Costar). Extracts from the cell-matrix constructs were
prepared by adding 10 mL ammonium acetate buffer and 0.5 mg/mL
Proteinase K to a test-tube containing a cell-matrix construct. The
mixture was incubated at 60.degree. C. overnight. After completion
of digestion, the mixture was spun down and the supernatant extract
was transferred to a separate tube for hyaluronic acid assay. A
96-well plate was coated with 50 .mu.L of 20 .mu.g/mL HA binding
protein in 0.1 M NaHCO.sub.3 solution and stored overnight at
4.degree. C. The plate was then washed three times with 0.85% NaCl
containing 0.05% Tween 20. To each well was then added 250 .mu.L
blocking solution (sodium phosphate buffer, 10 mmol pH=7.4
containing 3% BSA and 0.9% NaCl, PBS+3% BSA) and the plate was
incubated at RT for 2 h. The plate was then washed three times with
0.85% NaCl containing 0.05% Tween 20. To the plate was then added
50 .mu.L of standard HA solutions and extracts from both
experimental conditions, including various dilutions of these
conditions. The plate was incubated at room temperature (about
20.degree. C.) for 2 hours. The plate was then washed three times
with 0.85% NaCl containing 0.05% Tween 20 and to each well was
added 50 .mu.L of biotinylated HA (1:2000 dilution) and then
incubated for 2 hours at room temperature. The plate was then
washed three times with 0.85% NaCl containing 0.05% Tween 20 and
then added to each well was 50 .mu.L of HRP-avidin D (1:3000
dilution). The plate was then incubated for 45 minutes at room
temperature. The plate was then washed three times with 0.85% NaCl
containing 0.05% Tween 20 and to each well was added 100 .mu.L of
ortho-phenylenediamine substrate solution. The plate was incubated
at 37.degree. C. for 10 minutes. The reaction was stopped by
addition of 50 .mu.L of 1M HCl. Finally, using a plate reader, the
absorbance was read at 492 nm and recorded.
[0118] Absorbance measurements were averaged and converted to
quantity measures. Circular cell-matrix constructs (75 mm diameter)
formed in a serum containing media were determined to each contain
about 200 .mu.g hyaluronic acid while those formed in chemically
defined medium each contained about 1.5 mg hyaluronic acid.
Example 14
Physical Testing and Mechanical Properties of the Cell-Matrix
Construct Produced
[0119] The mechanical properties of the tissue constructs of
Example 1 (cell-matrix construct), Example 2 (cell-matrix construct
with a keratinocyte layer thereon), and Example 3 (cell-matrix
construct formed in defined medium) were quantified by membrane
inflation tests. These tests are similar to assays used clinically
(e.g. Dermaflex.RTM., Cyberderm Inc., Media, Pa., and
Cutameter.RTM., Courage Khazaka, Cologne, Germany) but involve
higher pressures including pressures able to burst the membrane.
The sample cell-matrix construct was laid flat on a polycarbonate
block centered over a cylindrical well 10 mm in diameter filled
with normotonic saline. A metal plate with a circular hole
corresponding to the diameter of the cylindrical well was placed
over the sample and clamped to the block. The samples were then
inflated by infusing additional saline into the well with a syringe
pump. The resulting pressure was measured with a pressure
transducer. Pressurization was canned out until device failure, the
burst strength, which averaged at 439.02 mm Hg for the cell-matrix
construct generated by the method of Example 1; 998.52 mm Hg for
the samples of the cell-matrix construct with a keratinocyte layer
generated by the method of Example 2; and, 1542.26 mm Hg for the
samples cell-matrix construct formed in defined medium generated
according to the method of Example 3.
[0120] To determine the thermal melting point of the dermal matrix,
samples (cell-matrix construct), taken at 21 days were prepared
using procedure described in Example 1. The samples denaturation
temperature was determined by analysis with Mettler Toledo
(Highston, N.J.) differential scanning calorimeter (DSC product
#DSC12E). For our purposes, the melting temperature was determined
by heating the sample from 45 and 80.degree. C. at a rate of
1.degree. C./minute. The average denaturation temperature for the
samples is 60.8.+-.1.2.degree. C. (n=3).
[0121] The suture retention and pull strength of the epidermalized
matrix created using the procedures in Examples 1 (cell-matrix
construct) and 3 (cell-matrix construct formed in defined medium)
were measured to determine the suturability of the construct in
certain clinical situations. Suture retention strength of the 21
day old human dermal matrix was determined using method described
in American National standards publication for Vascular Graft
Prosthesis (Instruments, 1986) using a Mini-Bionex 858 test system
(MTS systems Corporation, Minneapolis, Minn.)
[0122] For the samples of Example 1, (cell-matrix construct), the
tensile strength was determined to be 365 N/m; for samples prepared
according to Example 2 (cell-matrix construct with a keratinocyte
layer), the tensile strength was 2720 N/m.
[0123] The suture retention strength for samples prepared according
to Example 1 was 0.14 N; for those prepared according to Example 2,
0.22 N.
[0124] The constructs created as described in Examples 1, 2 and 3
have been made in both 24 mm and 75 mm diameters. The constructs
made by the culturing techniques of all 3 methods are cohesive
tissue-like structures are easily peeled form the membrane with
minimal force, hence "peelable", and able to be physically handled
and manipulated for use and testing without damage occurring.
Example 15
In Vitro Formation of a Collagenous Matrix by Human Neonatal
Foreskin Fibroblasts in Chemically Defined Medium
[0125] Human neonatal foreskin fibroblasts were expanded using the
procedure described in Example 1. Cells were then resuspended to a
concentration of 3.times.10.sup.6 cells/ml, and seeded on to 0.4
micron pore size, 24 mm diameter tissue culture treated membrane
inserts in a six-well tray at a density of 3.0.times.10.sup.6
cells/TW (6.6.times.10.sup.5 cells/cm.sup.2). Cells in this example
were cultured in chemically defined medium throughout.
[0126] The medium contained: a base 3:1 mixture of DMEM, Hams F-12
medium (Quality Biologics, Gaithersburg, Md.), 4 mM GlutaMAX (Gibco
BRL, Grand Island, N.Y.) and additives: 5 ng/ml human recombinant
epidermal growth factor (Upstate Biotechnology, Lake Placid, N.Y.),
1.times.10.sup.-4 M ethanolamine (Fluka, Ronkonkoma, N.Y. cat.
#02400 ACS grade), 1.times.10.sup.-4 M o-phosphoryl-ethanolamine
(Sigma, St. Louis, Mo.), 5 .mu.g/ml transferin (Sigma, St. Louis,
Mo.), 20 .rho.M triiodothyronine (Sigma, St. Louis, Mo.), and 6.78
ng/ml selenium (Sigma Aldrich Fine Chemicals Company, Milwaukee,
Wis.), 50 ng/ml L-ascorbic acid (WAKO Chemicals USA, Inc.), 0.2
.mu.g/ml L-proline (Sigma, St. Louis, Mo.), 0.1 .mu.g/ml glycine
(Sigma, St. Louis, Mo.).
[0127] To the basic medium above, other components were added in
these separate Conditions: [0128] 1. 5 .mu.g/ml insulin (Sigma, St.
Louis, Mo.), 0.4 .mu.g/ml hydrocortisone (Sigma, St. Louis, Mo.),
0.05% polyethylene glycol (PEG) (Sigma, St. Louis, Mo.). [0129] 2.
5 .mu.g/ml insulin (Sigma, St. Louis, Mo.), 0.4 .mu.g/ml
hydrocortisone (Sigma, St. Louis, Mo.). [0130] 3. 375 .mu.g/ml
insulin (Sigma, St. Louis, Mo.), 6 .mu.g/ml hydrocortisone (Sigma,
St. Louis, Mo.).
[0131] Samples were formalin fixed and processed for hemotoxylin
and eosin staining for light microscope analysis. Visual
histological evaluation demonstrated that the Condition 2 lacking
PEG demonstrated a comparably similar matrix as Condition 1
containing PEG. Biochemical analysis measuring the collagen content
of the construct showed nearly the same amount of collagen in both:
168.7.+-.7.98 .mu.g/cm.sup.2 for Condition 1 with PEG as compared
to 170.88.+-.9.07 .mu.g/cm.sup.2 for Condition 2 without PEG.
Condition 3 containing high levels of insulin and hydrocortisone
showed a higher expression of matrix, including collagen, at a
timepoint earlier than the other two conditions. Besides
endogenously produced fibrillar collagen, decorin and
glycosaminoglycan were also present in the cell-matrix constructs
in all Conditions. The cultured dermal construct formed by the
method of Condition 2 of this Example is shown in FIG. 2. Shown in
FIG. 2 is a photomicrograph of a fixed, paraffin embedded,
hematoxylin and eosin stained section of a cell-matrix construct
formed from cultured human dermal fibroblasts in chemically defined
medium at 21 days. The porous membrane appears as a thin
translucent band below the construct and it can be seen that the
cells grow on the surface of the membrane and do not envelope in
integrate the membrane with matrix.
[0132] FIG. 3 shows transmission electron microscope (TEM) images
of two magnifications of cultured dermal construct formed by the
method of Condition 2 of this Example at 21 days. FIG. 3A is a
7600.times. magnification showing alignment of endogenous collagen
fibers between the fibroblast. FIG. 3B is a 19000.times.
magnification of fuuy formed endogenous collagen fibers
demonstrating fibril arrangement and packing.
[0133] In all Conditions of this Example, the cultured dermal
constructs formed comprise dermal fibroblasts and endogenously
produced matrix. All have fully formed collagen fibrils in packed
organization arranged between the cells. Their fibrous qualities,
thickness, and cohesive integrity give the construct considerable
strength to allow it to be peelably removed from the culture
membrane and handled as it is transferred to a patient to be
treated with the construct, as in a graft or implant.
Example 16
Full Thickness Skin Construct
[0134] Using a 21 day dermal construct formed by human dermal
fibroblasts under chemically defined conditions according to the
method of Condition 2 (without PEG) described in Example 15, above,
normal human neonatal foreskin epidermal keratinocytes were seeded
on the top surface of the cell-matrix construct to form the
epidermal layer of the skin construct.
[0135] The medium was aseptically removed from the culture insert
and its surrounds. Normal human epidermal keratinocytes were scaled
up to passage 4 from frozen subculture cell stock to confluence.
Cells were then released from the culture dishes using
trypsin-versene, pooled, centrifuged to form a cell pellet,
resuspended in epidermalization medium, counted and seeded on top
of the membrane at a density of 4.5.times.10.sup.4 cells/cm.sup.2.
The constructs were then incubated for 90 minutes at
37.+-.1.degree. C., 10% CO.sub.2 to allow the keratinocytes to
attach. After the incubation, the constructs were submerged in
epidermalization medium. The epidermalization medium is composed
of: a 3:1 base mixture of Dulbecco's Modified Eagles Medium (DMEM)
(containing no glucose and no calcium, BioWhittaker, Walkersville,
Md.) and Hams F12 medium (Quality Biologics Gaithersburg, Md.),
supplemented with 0.4 .mu.g/ml hydrocortisone (Sigma St. Louis,
Mo.), 1.times.10.sup.-4 M ethanolamine (Fluka, Ronkonkoma, N.Y.),
1.times.10.sup.-4 M o-phosphoryl-ethanolamine (Sigma, St. Louis,
Mo.), 5 .mu.g/ml insulin (Sigma, St. Louis, Mo.), 5 .mu.g/ml
transferin (Sigma, St. Louis, Mo.), 20 .rho.M triiodothyronine
(Sigma, St. Louis, Mo.), 6.78 ng/ml selenium (Aldrich), 24.4
.mu.g/ml adenine (Sigma Aldrich Fine Chemicals Company, Milwaukee,
Wis.), 4 mM L-glutamine (BioWhittaker, Walkersville, Md.), 50
.mu.g/ml L-ascorbate sodium salt (Sigma Aldrich Fine Chemicals
Company, Milwaukee, Wis.), 16 .mu.M linoleic acid (Sigma, St.
Louis, Mo.), 1 .mu.M tocopherol Acetate (Sigma, St. Louis, Mo.) and
50 .mu.g/ml gentamicin sulfate (Amersham, Arlington Heights, Ill.).
The constructs were cultured in the epidermalization medium for 2
days at 37.+-.1.degree. C. 10.+-.1% CO.sub.2.
[0136] After 2 days the medium was exchanged with fresh medium
composed as above, and returned to the incubator set at
37.+-.1.degree. C., 10.+-.1% CO.sub.2 for 2 days. After the 2 days,
the carrier containing the construct was aseptically transferred to
new culturing trays with sufficient media to achieve a fluid level
just to the surface of the carrier membrane to maintain the
developing construct at the air-liquid interface. The air
contacting the top surface of the forming epidermal layer allows
stratification of the epithelial layer. The constructs were
incubated at 37.+-.1.degree. C., 10% CO.sub.2, and low humidity, in
media with media changes every 2-3 days for 7 days. This medium
contained a 1:1 mixture of Dulbecco's modified Eagle's medium
(DMEM) (containing no glucose and no calcium, BioWhittaker,
Walkersville, Md.), Hams F-12 medium (Quality Biologics,
Gaithersburg, Md.), supplemented with 0.4 .mu.g/ml hydrocortisone
(Sigma, St. Louis, Mo.), 5.times.10.sup.-4 M ethanolamine (Fluka,
Ronkonkoma, N.Y.), 5.times.10.sup.-4 M o-phosphoryl-ethanolamine
(Sigma, St. Louis, Mo.), 5 .mu.g/ml insulin (Sigma, St. Louis,
Mo.), 5 .mu.g/ml transferrin (Sigma, St. Louis, Mo.), 20 .rho.M
triiodothyronine (Sigma, St. Louis, Mo.), 6.78 ng/ml selenium
(Sigma Aldrich Fine Chemicals Company), 24.4 .mu.g/ml adenine
(Sigma Aldrich Fine Chemicals Company), 4 mM L-glutamine
(BioWhittaker, Walkersville, Md.), 2.65 .mu.g/ml calcium chloride
(Mallinckrodt, Chesterfield, Mo.), 16 .mu.M linoleic acid (Sigma,
St. Louis, Mo.), 1 .mu.M tocopherol acetate (Sigma, St. Louis,
Mo.), 1.25 mM serine (Sigma, St. Louis, Mo.), 0.64 mM choline
chloride (Sigma, St. Louis, Mo.) and 50 .mu.g/ml gentamicin sulfate
(Amersham, Arlington Heights, Ill.). The cultures were fed every
2-3 days, for 14 days.
[0137] Samples, in triplicate, were submitted 10, 12, and 14 days
after the construct was lifted to the air-liquid interface for
hematoxylin and eosin processing as described in Example 1 to
determine gross appearance under light microscopy. The resulting
construct was a bilayer skin construct consisted of a lower dermal
layer consisting of dermal fibroblasts surrounded by matrix
overlaid by an upper epidermal layer of stratified and
differentiated keratinocytes. The bilayer skin construct of this
Example is shown in FIG. 4. FIG. 4 is a photomicrograph of a fixed,
paraffin embedded, hematoxylin and eosin stained section of a
cultured skin construct formed in chemically defined media in the
absence of exogenous matrix components comprising a cell-matrix
construct formed from cultured human dermal fibroblasts in
chemically defined medium with a multilayered, differentiated
epidermis formed from cultured human keratinocytes in chemically
defined medium.
Example 17
Formation of a Collagenous Matrix by Human Buccal Fibroblasts
[0138] The purpose of this experiment is to produce a cell-matrix
construct from buccal fibroblasts isolated from human cheek tissue.
Buccal were cultured in T-150 flasks in DMEM containing 10% NBCS
medium After 7 days, to expand the number of cells further, buccal
cells were harvested and passaged into nine T-150 flasks at
4.0.times.10.sup.6 cells in DMEM containing 10% NBCS medium and
cultured until confluence at which time the cells were
harvested.
[0139] To harvest the cells, the medium was aspirated from the
culture flasks. To rinse the monolayer, sterile-filtered phosphate
buffered saline was added to the bottom of each culture flask and
then aspirated from the flasks. Cells were released from the flask
by adding 5 mL trypsin-versene glutamine (BioWhittaker,
Walkersville, Md.) to each flask and gently rocking to ensure
complete coverage of the monolayer. Cultures were returned to the
incubator. As soon as the cells were released 5 ml of SBTI (Soybean
Trypsin Inhibitor) was added to each flask and mixed with the
suspension to stop the action of the trypsin-versene. The cell
suspension was removed from the flasks and evenly divided between
sterile, conical centrifuge tubes. Cells were collected by
centrifugation at approximately 800-1000.times.g for 5 minutes.
[0140] Cells were resuspended using fresh medium to a concentration
of 3.0.times.10.sup.6 cells/ml, and seeded onto 0.4 micron pore
size, 24 mm diameter tissue culture treated inserts
(TRANSWELL.RTM., Corning Costar) in a six-well tray at a density of
3.0.times.10.sup.6 cells/insert (6.6.times.10.sup.5
cells/cm.sup.2). The cells were maintained in an incubator at
37.+-.1.degree. C. with an atmosphere of 10.+-.1% CO.sub.2 and fed
medium containing: a base 3:1 mixture of DMEM, Hams F12 medium
(Quality Biologics, Gaithersburg, Md.), 4 mM GlutaMAX (Gibco BRL,
Grand Island, N.Y.) and additives: 5 ng/ml human recombinant
epidermal growth factor (Upstate Biotechnology, Lake Placid, N.Y.),
0.4 .mu.g/ml hydrocortisone (Sigma, St. Louis, Mo.),
1.times.10.sup.-4 M ethanolamine (Fluka, Ronkonkoma, N.Y. cat.
#02400 ACS grade), 1.times.10.sup.-4 M o-phosphoryl-ethanolamine
(Sigma, St. Louis, Mo.), 5 .mu.g/ml insulin (Sigma, St. Louis,
Mo.), 5 .mu.g/ml transferrin (Sigma, St. Louis, Mo.), 20 .rho.M
triiodothyronine (Sigma, St. Louis, Mo.), and 6.78 ng/ml selenium
(Sigma Aldrich Fine Chemicals Company, Milwaukee, Wis.), 50 ng/ml
L-ascorbic acid (WAKO Chemicals USA, Inc.), 0.2 .mu.g/ml L-proline
(Sigma, St. Louis, Mo.), 0.1 .mu.g/ml glycine (Sigma, St. Louis,
Mo.) and 0.05% poly-ethylene glycol (PEG) (Sigma, St. Louis,
Mo.).
[0141] At day 1 post seeding, medium was replaced with Serum Free
Production Media, exchanged every 2-3 days for 21 days. At day 21,
samples were fixed in formalin for histology. Three samples were
used for protein and collagen production analysis.
[0142] Collagen production for 24 mm diameter constructs averaged
519 .mu.g per construct after 21 days in culture. Total protein
production for 24 mm diameter constructs averaged 210 .mu.g per
construct after 21 days in culture. Morphologically, the buccal
fibroblast cell-matrix construct, a cultured tissue construct of
oral connective tissue, showed buccal fibroblasts surrounded by
matrix while physically, the construct had physical bulk and
integrity.
[0143] Although the foregoing invention has been described in some
detail by way of illustration and Examples for purposes of clarity
and understanding, it will be obvious to one of skill in the art
that certain changes and modifications may be practiced within the
scope of the appended claims.
* * * * *