U.S. patent application number 11/263321 was filed with the patent office on 2006-12-28 for epidermal and dermal equivalents.
This patent application is currently assigned to DFB Pharmaceuticals, Inc.. Invention is credited to Thomas Hunziker, Alain Limat.
Application Number | 20060292126 11/263321 |
Document ID | / |
Family ID | 26999963 |
Filed Date | 2006-12-28 |
United States Patent
Application |
20060292126 |
Kind Code |
A1 |
Hunziker; Thomas ; et
al. |
December 28, 2006 |
Epidermal and dermal equivalents
Abstract
The present invention relates to the treatment of skin defects
by organotypically-cultured autologous keratinocytes isolated from
the outer root sheath of anagen or growing hair. Methods for
primary, as well as subsequent organotypic cultures (i.e.,
epidermal equivalents) in fully-defined media supplemented by
autologous human serum and substances isolated form blood
components, with minimal allogeneic biological supplements, are
disclosed herein. Techniques to prepare epidermal equivalents for
transplantation by use of a biocompatible glue are also disclosed
herein.
Inventors: |
Hunziker; Thomas;
(Oberhofen, CH) ; Limat; Alain; (Tavel,
CH) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE.
SUITE 2400
AUSTIN
TX
78701
US
|
Assignee: |
DFB Pharmaceuticals, Inc.
San Antonio
TX
|
Family ID: |
26999963 |
Appl. No.: |
11/263321 |
Filed: |
October 31, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10031188 |
May 13, 2002 |
7014849 |
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PCT/IB00/01076 |
Jul 20, 2000 |
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11263321 |
Oct 31, 2005 |
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09546269 |
Apr 10, 2000 |
6730513 |
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10031188 |
May 13, 2002 |
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09358181 |
Jul 20, 1999 |
6548058 |
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10031188 |
May 13, 2002 |
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Current U.S.
Class: |
424/93.7 ;
435/371 |
Current CPC
Class: |
C12N 5/0628 20130101;
A61L 27/60 20130101; C12N 2533/30 20130101; C12N 2510/04 20130101;
C12N 5/0627 20130101; C12N 5/063 20130101; A61L 27/3804 20130101;
A61P 17/00 20180101; A61P 17/02 20180101; A61L 2430/18
20130101 |
Class at
Publication: |
424/093.7 ;
435/371 |
International
Class: |
A61K 35/36 20060101
A61K035/36; C12N 5/08 20060101 C12N005/08 |
Claims
1-71. (canceled)
72. A method for preparing an epidermal or complex skin equivalent
suitable for subsequent treatment of a skin defect, said method
comprising: a) culturing an anagenic hair follicle to obtain outer
root sheath cells; b) culturing said outer root sheath cells to
obtain keratinocyte precursor cells; and c) preparing an epidermal
or complex skin equivalent comprising said keratinocyte precursor
cells, wherein said hair follicle is intact.
73. The method of claim 72, wherein said culturing steps (a) and
(b) are performed in a medium containing human serum at a
concentration less than 5%.
74. The method of claim 72, wherein said keratinocyte precursor
cells are seeded at a density of between 3.times.10.sup.4
cells/cm.sup.2 and 1.times.10.sup.5 cells/cm.sup.2.
75. The method of claim 72, wherein said keratinocyte precursor
cells are selected by: d) primary-culturing said outer root
sheath-derived keratinocyte precursor cells by adhering said intact
anagenic hair to a microporous membrane, said membrane possessing
growth-arrested/limited feeder cells on its undersurface so as to
select for keratinocyte precursor cells from the outer root sheath
of hair; e) organotypically-culturing the outer root sheath cells
harvested from said primary cultures by modulating a microporous
membrane which also possesses growth-arrested limited feeder cells
on its undersurface; and f) generating said epidermal or complex
skin equivalent for subsequent use as a graft insert by placing a
carrier membrane on top of said organotypic-culture from step d)
and detaching said complex skin or epidermal equivalent; whereby
said graft comprises the keratinocyte precursor cells and carrier
membrane as a single laminar unit, said keratinocyte precursor
cells being seeded on said carrier membrane at a density of between
3.times.10.sup.4 cells/cm.sup.2 and 1.times.10.sup.5
cells/cm.sup.2.
76. The method of claim 75, wherein the culture density of said
growth-arrested/limited feeder cells on said microporous membrane
is between about 1.times.10.sup.4 cells/cm.sup.2 and about
5.times.10.sup.4 cells/cm.sup.2.
77. The method of claim 75, wherein said growth-arrested/limited
feeder cells are banked or immortalized cells.
78. The method of claim 75, wherein said epidermal or complex skin
equivalent comprises outer root sheath cells cultured in a medium
containing only homologous or autologous releasates from blood
components.
79. The method of claim 78, wherein said epidermal or complex skin
equivalent comprises outer root sheath cells cultured in a medium
containing only homologous or autologous releasates from blood
components at a concentration of about 0.1% to about 20%.
80. The method of claim 75, wherein said microporous membrane is
coated by one or more extracellular matrix substances selected from
the group consisting off fibrin, fibronectin, collagens, laminins
and hyaluronan.
81. The method of claim 75, wherein said microposous membrane
possesses a growth-arrested/limited feeder cells system on its
undersurface with said feeder cells of at least one type of cell
selected from the group consisting of human dermal fibroblasts,
epidermal cells, mesenchymal cells, neuronal cells and endothelial
cells.
82. The method of claim 75, wherein said carrier membrane is made
from one or more types of materials selected from the group
consisting of polyester, PTFE, polyurethane, hyaluronic acid,
polyactic acid, collagen and a silicone or Vasiline gauze
dressing.
83. The method of claim 75, wherein the size of said epidermal or
complex skin equivalent is selected from the group consisting of
1.0 cm, 1.5 cm, 2.0 cm and 2.5 cm in diameter.
84. The method of claim 72, wherein said epidermal or complex skin
equivalent is coated on its top or cornified side with a fibrin
glue.
85. The method of claim 84, wherein said fibrin glue comprises one
or more antimicrobial, anti-fungal or anti-viral agents emulsified
therein.
86. The method of claim 72, wherein said outer root sheath cells
are homologous cells.
87. The method of claim 72, wherein said epidermal or complex skin
equivalent comprises outer root sheath cells cultured in a medium
containing only homologous or autologous biological
supplements.
88. The method of claim 72, wherein said epidermal equivalent is
coated on its top or cornified side with a carrier membrane.
89. The method of claim 72, wherein said outer root sheath cells
are autologous cells obtained from an individual who will
subsequently undergo treatment for a skin defect.
90. The method of claim 72, further comprising shipping or
transporting said epidermal or complex skin equivalent by:
detaching said epidermal or complex skin equivalent from said
culture medium; transferring said equivalent onto a carrier; and
contacting said epidermal equivalent and carrier with a solidified
or gelled medium.
91. The method of claim 90, wherein said equivalent is coated on
its top or cornified side with a carrier membrane.
92. The method of claim 91, wherein said equivalent is further
sealed and shipped for future use in grafting.
93. The method of claim 90, wherein said solidified or gelled
medium is selected from the group consisting of agarose, methyl
cellulose and another gelifying substance.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the field of cell culture of human
keratinocyte precursor and dermal fibroblast cells. The invention
also relates to the use of cultured keratinocyte precursor cells in
the repair of skin defects by skin grafting procedures.
BACKGROUND OF THE INVENTION
[0002] The healing of skin defects progresses through three general
phases: (i) inflammation, (ii) wound cell migration and mitosis,
and (iii) extracellular matrix production and remodeling. The
ordered sequence of these events is thought to be orchestrated by
interactions among cells, growth factors, and extracellular matrix
proteins. A crucial step of skin wound healing is epidermal
regeneration (i.e., re-epithelialization). Besides interfollicular
epidermal keratinocytes from the wound edges, the outer root sheath
(ORS) cells from residual hair follicles also contribute to this
process (see e.g., Eisen et al., 15 J. Invest. Dermatol. 145-155
(1955)). The ORS of hair follicles is comprised largely of
undifferentiated keratinocytes that encompass the cylindrical
structures of the hardened inner root sheath and the hair shaft
(see e.g., Montagna & Parakkal, In: The Structure and Function
of Skin 172-258 (Academic Press New York, N.Y., 1974)). Recent
literature has also indicated that ORS cells are at a lower level
of commitment to differentiation than the basal interfollicular
keratinocytes (see e.g., Coulombe et al., 109 J. Cell Biol.
2295-2312 (1989); Limat et al., 194 Exp. Cell Res. 218-227 (1991);
Limat et al., 275 Cell Tissue Res. 169-176 (1994)), and
label-retaining cells have been detected in the animal as well as
the human ORS region near the bulge area which possibly represent
stem cells for skin epithelial tissues (see e.g., Cotsarelis et
al., 61 Cell 1329-1337 (1990); Kobayashi et al., 90 Proc. Nat.
Acad. Sci. USA 7391-7395 (1993); Yang et al., 105 J. Invest.
Dermatol. 14-21 (1993); Rochat et al., 76 Cell 1073-1076 (1994);
Moll, 105 J. Invest. Dermatol. 14-21 (1995)). Additionally, human
ORS cells which are isolated from plucked anagen scalp hair
follicles can be expanded extensively in vitro (see e.g., Weterings
et al., 104 Brit. J. Dermatol. 1-5 (1981); Limat & Noser, 87 J.
Invest. Dermatol. 485-488 (1986); Imcke et al., 17 J. Am. Acad.
Dermatol. 779-786 (1987); Limat et al., 92 J. Invest. Dermatol.
758-762 (1989)). Under conventional submerged culture conditions,
ORS cells resemble interfollicular epidermal keratinocytes by both
morphologic and biochemical (e.g., keratin profiles) criteria (see
e.g., Stark et al., 35 Differentiation 236-248 (1987); Limat et
al., 92 J. Invest. Dermatol. 758-762 (1989); Limat et al., 642 Ann.
N.Y. Acad. Sci. 125-147 (1991)). In organotypic co-cultures with
human dermal fibroblasts (i.e., under conditions mimicking the
epidermal environment), ORS cells with respect to histological,
immunohistological, ultrastructural and biochemical criteria
develop a stratified epithelium reminiscent of regenerating
epidermis (see e.g., Lenoir et al., 130 Dev. Biol. 610-620 (1988);
Limat et al., 194 Exp. Cell Res. 218-227 (1991); Limat et al., 642
Ann. N.Y. Acad. Sci. 125-147 (1991)). If such organotypic cultures
are grafted onto nude mice, ORS cells form a regular neo-epidermis
that is under homeostatic control (see e.g., Limat et al. 59
Transplantation 1032-1038 (1995)). Thus, human ORS cells are of
considerable interest for clinical application.
[0003] In the previous decade, interest has focused on the use of
cultured epithelial cells for wound coverage. First, sheets of
cultured autologous interfollicular keratinocytes were grafted
successfully on acute wounds, mainly in the treatment of larger
third degree burns (see e.g., O'Connor et al., 1 Lancet 75-78
(1981); Compton et al., 60 Lab. Invest. 600-612 (1989)) but also of
epidermolysis bullosa (see e.g., Carter et al., 17 J. Am. Acad.
Dermatol. 246-250 (1987)), pyoderma gangrenosum (see e.g., Dean et
al., 26 Ann. Plast. Surg. 194-195 (1991); Limova & Mauro. 20 J.
Dermatol. Surg. Oncol. 833-836 (1994)), and wounds after excision
of giant congenital nevi (see e.g., Gallico et al., 84 J. Plast.
Reconstr. Surg. 1-9 (1989)) or separation of conjoined twins (see
e.g., Higgins et al., 87 J. Royal Soc. Med. 108-109 (1994)).
[0004] In contrast to the treatment of such acute wounds, the
grafting of chronic wounds (e.g.. leg ulcers) with cultured
keratinocytes has been much less successful. Allografts do not
result in a permanent "take" (see e.g., Fabre. 29 Immunol. Lett.
161-166 (1991)) and thus may be classified as a "quite effective
but expensive biological dressing" (see Phillips et al., 21 J. Am.
Acad. Dermatol. 191-199 (1989). A reproducible major definite
"take" of autologous keratinocyte grafted by various modalities
including sheets of submerged keratinocyte cultures consisting of
only a few, noncornified cell layers (Hetton et al., 14 J. Am.
Acad. Dermatol. 399-405 (1986); Leigh & Purkis, 11 Clin. Exp.
Dernatol. 650-652 (1986); Leigh et al., 117 Brit. J. Dermatol.
591-597 (1987); Harris et al., 18 Clin. Exp. Dermatol. 417-420
(1993)), trypsinized single cells attached to collagen-coated
dressings (Brysk et al., 25 J. Am. Acad. Dermatol. 238-244 (1991)),
skin equivalents (Mol et al., 24 J. Am. Acad. Dermatol. 77-82
(1991)) has yet to be convincingly documented within the scientific
literature. The same lack of quantitative findings also holds true
for various reports on the grafting of freshly isolated, autologous
interfollicular keratinocytes (Hunyadi et al., 14 J. Dermatol.
Surg. Oncol. 75-78 (1988)) or ORS cells (Moll et al., 46 Hautarzt
548-552 (1995)) fixed to the wound bed by the use of a fibrin glue.
However, it should be noted that the disadvantages of the bovine
serum used during cultivation of the keratinocytes may contribute
to reduced "take" rate, due to the fact that it resists in
keratinocytes (see e.g., Johnson et al., 11 J. Burn Care Rehab.
504-509 (1990)).
SUMMARY OF THE INVENTION
[0005] Prior to the disclosure of the present invention herein, the
standard methodology for the generation of a primary culture of ORS
keratinocytes consisted of the plucking of an anagen (i.e., growing
hair shaft) hair followed by a careful microscopic dissection to
remove the hair bulbs and the infundibular hair shaft. The
resulting outer root sheath was then placed on the culture insert
for initiation of the primary keratinocyte culture. However,
numerous subsequent studies (approximately 200), wherein the anagen
hair was placed directly on the culture insert without performing
the initial micro-dissection to remove the hair bulbs and the
infundibular hair shaft, have demonstrated that such tedious and
time-consuming dissection of the plucked anagen hair was not
required. This has served to markedly simplify the handling
process, reduce the risk for contamination, and resulted in more
efficient initiation of keratinocyte cell plating.
[0006] Accordingly, it is an object of the present invention to
provide improved and simplified methods for the generation of
keratinocytes or keratinocyte precursors from outer root sheath
cells (ORS cells) in fully defined culture conditions for the
treatment of various types of skin defects (e.g., chronic wounds
such as leg ulcers, diabetic ulcers, pressure sores, and the like)
in both humans and animals. In addition to their use in the
treatment of wounds, keratinocytes may also be used in plastic and
cosmetic surgery, or whenever there is a demand for such skin
support (e.g., post operative following the removal of tattoos,
naevi, skin cancer, papillomas, after amputation, in sex
transformation or re-virgination, rejuvenation of actinically
damaged skin after skin resurfacing, tympanoplasty,
epithelialization of external ear canal, and the like).
[0007] These aforementioned objectives are accomplished by
explantation and culture of plucked, anagen or growing hairs in
toto upon microporous membranes carrying human fibroblast feeder
cells at their under-surface. In such primary cultures, large
numbers of ORS cells can be easily and repeatedly obtained,
irrespective of the donor's chronological age. Such ORS cells may
be used for the subsequent preparation of complex skin, i.e.,
dermo-epidermal, or epidermal equivalents or kept frozen and stored
in order to use them at a later time point.
[0008] The subsequent preparation of skin or epidermal equivalents
is achieved by the "seeding" of these ORS cells upon a modified,
microporous membrane carrying fibroblast feeder cells (most
preferably growth-arrested/limited human dermal fibroblast "feeder
cells") at their under-surface. During culture, these ORS cells
undergo tissue differentiation which has been demonstrated to be
similar to that of normal epidermis. This finding is most probably
due to a large compartment of proliferating cells. The modified
culture conditions which are disclosed herein are important for the
successful treatment of chronic wounds with epidermal equivalents
generated in vitro from autologous ORS cells.
[0009] A further object of the present invention is to provide
improved culture systems for ORS-derived keratinocytes by adhering
the anagenic hair onto a polymeric microporous membrane coated with
one or more molecules of extracellular matrix origin. These
improved cultures of ORS cells, designated as skin equivalents or
epidermal equivalents, may be used to treat skin defects,
especially chronic wounds.
[0010] Yet another object of the present invention is to produce
skin or epidermal equivalents using a reduced concentration of
allogenic or homologous serum. This greatly mitigates the risk of
disease transmission, for example, by clinical use of blood
products, by the use of autologous or homologous human serum and
substances derived or released from blood components (e.g., blood
platelets) for supplements in in vitro culturing steps.
[0011] A further object of the present invention is a methodology
which reduces the probability of mechanical damage (e.g.,
separation of the various constituent layers) of the skin or
epidermal equivalents during transport prior to
transplantation.
[0012] The clinical advantages of the methodology of the present
invention, as compared to grafting techniques of chronic wounds
which have been previously utilized, include, but are not limited
to: noninvasiveness (so that the cells are available repeatedly),
the lack of need for surgical facilities or anesthesia during the
grafting procedure, and a short immobilization period of only 2
hours required following the grafting procedure.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice of the present invention, the
preferred methods and materials are now described. All publications
mentioned herein are incorporated herein in their entirety by
reference.
[0014] The term "keratinocyte layer" as used herein means an in
vitro generated keratinocyte tissue culture with more or less
differentiated structure. The term "epidermal equivalent" as used
herein means an in vitro generated organotypic tissue culture
resembling in its histological structure the natural epidermis
especially concerning the stratification and development of the
horny layer. A normal stratified epidermis consists of a basal
layer of small cuboidal cells, several spinous layers of
progressively flattened cells, a prominent granular layer and an
orthokeratotic horny layer. All these layers can be detected in the
epidermal equivalents that are subject of the invention.
Localization of those epidermal differentiation products that have
been assayed by immunohistochemistry (e.g. keratins, involucrin,
filaggrin, integrins) is similar to that found in normal
epidermis.
[0015] The term "autologous" as used herein means: (i) that
biological material to be transplanted is derived from the
individual to be treated with epidermal equivalents; or (ii) that
biological material added to tissue cultures comes from the donor
of the cells for tissue culture.
[0016] The term "homologous" as used herein means: (i) that
biological material to be transplanted is derived from one or more
individuals of the same species as the individual to be treated
with epidermal equivalents; or (ii) that biological material added
to tissue cultures comes from one or more individuals of the same
species as the donor of cells for the tissue culture.
[0017] The term "organotypic culture" and the like, refers to
culture of cells under conditions that promote differentiation of
the cells. Under conditions of organotypic culture, proliferation
of the cells is slowed compared to culture under "proliferative"
conditions such as primary culture conditions, and may be
completely stopped. In the present case, an important condition for
organotypic culture is maintenance of the cells at the air-liquid
interface, a so-called "lifted" culturing condition.
[0018] The term "releasate from blood components" (e.g., blood
platelets) as used herein means any combination of cytokines or
other growth factors obtained from blood components (e.g., blood
platelets). Platelets stimulated with, for example, thrombin
release the content of their alpha granules into the surrounding
medium. Alpha granules usually contain several cytokines (e.g.,
platelet derived growth factor (PDGF), epidermal growth factor
(EGF), transforming growth factors alpha and beta (TGF alpha/beta),
platelet factor 4 (PF-4), platelet basic protein (PBP)). However,
it is possible to obtain cytokines and other growth factors from
platelets by other methods than stimulating with thrombin.
Moreover, other blood components produce growth factors and
cytokines as well. Monocytes, for example, produce IL-1, TNF alpha.
IL-6 and other substances of interest.
[0019] General Method for Preparing Epidermal Equivalents from ORS
Cells. Keratinocyte precursor cells are selected from outer root
sheath (ORS) of anagen or growing hair which is derived from the
individual which is to be subsequently treated with epidermal
equivalents. In general, approximately 40 hair follicles are
plucked from the scalp, and those in the anagen phase (i.e., a
growing hair shaft) are then selected under the dissecting
microscope. A total of four weeks of culture is usually required in
order to obtain approximately 1 cm.sup.2 of epidermal equivalents
from five hair follicles. However, with improved culture and
fermentation techniques it may be possible to get a higher yield
(i.e., a larger area of epidermal equivalents, within this period
of time).
[0020] The previous standard method for the generation of a primary
culture of ORS keratinocytes consisted of the plucking of an anagen
(i.e., growing hair shaft) hair followed by a careful microscopic
dissection to remove the hair bulbs and the infundibular hair
shaft. The resulting outer root sheath (ORS) was then placed on the
culture insert for initiation of the primary keratinocyte culture.
However, numerous subsequent studies (approximately 200), wherein
the anagen hair was placed directly on the culture insert without
performing the initial micro-dissection to remove the hair bulbs
and the infundibular hair shaft, have demonstrated that such
tedious and time-consuming dissection of the plucked anagen hair
was not required. This has served to markedly simplify the handling
process, reduce the risk for contamination, and resulted in more
efficient initiation of keratinocyte cell plating.
[0021] The selected anagen hairs were incubated in an appropriate
rinsing buffer containing various anti-microbial and anti-fungal
agents (e.g., fungizone, penicillin. and streptomycin). Following
this procedure, the entire plucked anagen hair is placed directly
on the culture insert and allowed to grow for several days,
preferably 7-14, days, and more preferably 8 to 10 days. An
optional, additional step is comprised of passaging the primary
culture and performing a secondary culture in order to obtain more
cellular material for the preparation of larger areas of epidermal
equivalents.
[0022] The culture insert, a microporous membrane coated with one
or more extracellular matrix substances (e.g., fibrin, fibronectin,
collagens, laminins or hyaluronan or mixtures thereof), carries a
growth-arrested/limited feeder cell system on its undersurface. The
coating of the membrane insert with such extracellular matrix
substances provides for: (i) an enhanced culture surface for the
initial attachment of the anagen hair (i.e.. it sticks easily and
remains stationary): (ii) a surface which significantly enhances
the migration of the ORS keratinocytes away from the outer root
sheath (ORS) anagen hair follicles; and (iii) increased growth
rates of the spreading ORS keratinocytes (i.e., the overall culture
time needed for production of fully differentiated skin or
epidermal equivalents) can be reduced to three weeks, instead of
four.
[0023] The aforementioned growth-arrested/limited feeder cell
system located on the under surface of the microporous insert
membrane is comprised of primary dermal fibroblasts obtained from a
human skin biopsy. The primary dermal fibroblasts are treated with
mitomycin-C for 4 to 6 hours prior to their use as a "feeder cell
layer" for the plucked anagen hair and then plated on the underside
of the culture insert. Growth arrest/limitation is induced by
either mitomycin-C or X-ray treatment or, preferably, the reduced
serum concentration below 5%, and preferably 2%. It should be noted
that, although some cultures had been performed using 10% fetal
calf serum (FCS; Boehringer Mannheim, Germany), the current
utilization of human serum, in order to reduce the number of
allogeneic ingredients, was found to provide markedly superior
outgrowth and proliferation of the ORS cells. Moreover, the human
serum is preferably utilized in a concentration of less than 5%,
and more preferably in a concentration of 2%. In the presence of
such low serum concentrations, the primary human dermal fibroblasts
of the present invention will become significantly, or completely
growth arrested. Hence, in this manner, two expensive and
potentially complicating steps in the autologous ORS culture system
may be removed. The two complicating steps include: (i) removal of
high serum >5% concentrations, which reduces the overall cost of
the process significantly and; (ii) the removal of mitomycin-C
treatment, which provides a fully mitomycin-C-free culture system
and eliminates any concerns regarding the total elimination of the
drug from the primary culture inserts prior to the growth of the
epidermal equivalents. In addition, the use of reduced serum
concentrations allows the alternative feeder cell-arresting
procedure (i.e., the X-ray exposure step) to be eliminated, thus
saving significant time and expense in the overall procedure.
[0024] Following expansion of the ORS cells to an appropriate
density (i.e., 1.times.10.sup.3 to 1.times.10.sup.6 cells/cm.sup.2,
and preferably 5.times.10.sup.4 to 1.times.10.sup.5
cells/cm.sup.2), they are used for preparation of epidermal
equivalents. Preferably, the cells are grown to confluence. The
epidermal equivalents are prepared by seeding ORS cells at an
appropriate cell density (i.e.. 30.times.10.sup.3 to 100.times.10
cells/cm.sup.2, and preferably 60.times.10.sup.3 cells/cm.sup.2)
within a culture device which is suitable for "lifting" the cells
up to the air-liquid interface during culture. Subsequently, one to
four days after seeding (preferably 3 days after seeding), the ORS
cells are exposed to air (e.g., by aspiration of the medium inside
the insert) and the cultures are then continued for approximately
10-20 days, and preferably for 14-18 days, in such "lifted" culture
condition. The medium is changed periodically during the lifted
culture; preferably every two to three days.
[0025] The present invention also encompasses skin equivalents
which include additional layers, and so are more complex structures
than epidermal equivalents. Skin equivalents comprise
differentiated ORS cells as their epidermal part and also a layer
comprising a matrix component, preferably one containing embedded
dermal fibroblasts and/or other cells (i.e., an "embedding
matrix"). Skin equivalents are made by placing a matrix with one or
more extracellular matrix substances (e.g., fibrin, fibronectin,
collagens, laminins or hyaluronan or mixtures thereof) on the upper
surface of the microporous membrane described above. When embedding
human dermal fibroblasts, preferably autologous human dermal
fibroblasts, the cells are embedded at a density of
1.times.10.sup.3 to 1.times.10.sup.7 cells/cm.sup.3; preferably
1.times.10.sup.4 to 1.times.10.sup.5 cells/cm.sup.3; and most
preferably approximately 5.times.10.sup.4 cells/cm.sup.3. The
primary culture of ORS cells is then seeded on top of the matrix
(preferably containing embedded dermal fibroblasts and/or other
cells) and organotypic culturing is performed as described above.
For a detailed description of the preparation of dermal equivalents
(see e.g., Limat et al., 194 Exp. Cell Res. 218-277 (1991)).
[0026] It should be noted, however, that the cells which are
embedded in the matrix need not be limited exclusively to dermal
fibroblasts; as epidermal, mesenchymal. neuronal and/or endothelial
cells can also be utilized. The embedded cells are preferably
obtained from skin tissue, are more preferably allogeneic cells,
and are most preferably autologous cells.
[0027] All culture steps are performed in an appropriate medium
which allows the proliferation of the ORS cells and their outgrowth
from the hair follicles, the medium is typically changed every 2 to
3 days. Generally, the medium utilized for all steps is the same.
The medium is typically based on a minimal medium and contains
several additional ingredients. One common ingredient is serum in a
concentration of 0.5-60%. In the preferred embodiment of the
present invention, human serum is used at a concentration of less
than 5%, and most preferably at a concentration of 2%. Furthermore,
with the development of serum-free media, it may be possible to
omit serum in toto. Epidermal growth factor (EGF) stimulates
migration of keratinocytes and delays their senescence which
results in stimulation of proliferation. Cholera toxin,
hydrocortisone, insulin, adenine and triiodothyronine have an
effect of stimulating proliferation. All of these ingredients are
thus useful in a medium for preparing epidermal equivalents.
Nevertheless, it may be possible to omit or replace one or another
of these ingredients.
[0028] Releasate from blood components (e.g., blood platelets,
monocytes or lymphocytes), may serve as a source of cell
proliferating activities, and therefore may substitute serum and
provide other above mentioned ingredients. For certain culture
periods the serum-containing medium might possibly be replaced by a
defined, serum-free medium, for example, SFM (Gibco Europe,
Ettlingen). The releasate from blood components (e.g., blood
platelets, monocytes or lymphocytes), especially of homologous or
autologous origin, may serve as a source of cell proliferating
activities and therefore may substitute serum and provide other
above mentioned ingredients or indeed may provide additional
ingredients. The blood components should be added to the culture
medium in a concentration of 0.1% to 20%, and preferably 1% to 5%,
after the releasate is brought-to the same final volume as the
blood from which these components are obtained. These releasates
contain several growth factors that are present in serum (e.g..
PDGF, ECF or TGFs). However, serum as well as releasates contain
many substances, and not all are characterized.
[0029] Releasate from blood platelets is obtained by centrifugation
of anti-coagulated whole blood, preferably human blood, in order to
pellet all cells except thrombocytes. The supernatant is
centrifuged once more to spin down the thrombocytes. The
thrombocytes are suspended in an appropriate buffer, e.g. phosphate
buffer and treated with thrombin in order to release their alpha
granules which contain a mixture of various growth factors (e.g.,
PDGF, PF-4, TGF-.beta., EGE, .beta.-thromboglobulin). In a further
centrifugation step all cellular material is removed. Finally, the
supernatant is supplemented with buffer to the volume of the
original blood sample from which the components are obtained. The
blood components should be added to the culture medium in a
concentration of 0.1% to 20%; preferably 1% to 10%; and more
preferably 2 to 5%.
[0030] Similarly, releasates can be obtained from other blood
cells, such as monocytes, by breaking up the cells (e.g., by
sonication, freeze-thaw method, or the like) and purifying the
growth factors (e.g., by filtration or immunological methods).
[0031] The blood component releasates can also be used to condition
the wound bed in the course of grafting the epidermal or dermal
equivalents. Furthermore, the culture medium containing the
releasates and used to perform the organotypic culturing step,
after having been conditioned by the cells, can be used to
condition the bed of the skin defect in the course of grafting the
epidermal or dermal equivalents.
[0032] Cultivation usually is performed in inserts with microporous
membranes, which contain homologous or autologous human dermal
fibroblasts (HDF), especially postmitotic HDF at their
undersurface. HDF secrete factors that condition the medium in
order to get a better growth of the epidermal equivalents. The HDF
layer can be formed from between 5.times.10.sup.3 to
1.times.10.sup.5 cells/cm.sup.2, and preferably approximately
1.times.10.sup.4 to 5.times.10.sup.4 cells/cm.sup.2. The HDF are
preferably postmitotic, but earlier passage cells can be used if
they are irradiated, treated with mitomycin-C, or otherwise treated
to inhibit their proliferation but maintain their metabolism, i.e.,
by reduction of serum concentration.
[0033] In one embodiment, the graft thickness for the complex
dermal ("complex skin") equivalents does not exceed 0.4 mm.
[0034] Microporous membranes are suitable as a culture substrate,
because they allow substances to diffuse from one side to the
other, but work as a barrier for cells. The pore size of the
membrane is not a limitation on the present invention, but should
be adequate so as to allow diffusion of proteins of up to 100,000
Daltons molecular weight, and preferably of up to 70,000 Daltons
molecular weight. The membrane should at least allow diffusion of
small hormones such as insulin, and allow passage of proteins of up
to 15,000 Daltons molecular weight. Other means than a microporous
membrane for performing the function of allowing diffusion of
soluble factors to the cultured ORS cells, while preventing mixing
of the ORS cells with the HDF would also be usable.
[0035] The microporous membranes typical in the art are usually
used. However, membranes fabricated from a biodegradable material
(e.g., polyhyaluronic acid or polylactic acid) can also be used.
When a biodegradable microporous membrane is employed it is
contemplated that the entire culture, including the differentiated
ORS cells, the microporous membrane and the HDF, will be
transplanted into the skin defect. Thus, in this alternative
embodiment, the HDF grown on the underside of the membrane need not
be post-mitotic or treated to preclude proliferation. While HDF
tend to be less immunogenic than keratinocytes, it is preferable
that when this embodiment is employed, the HDF be allogeneic cells,
preferably autologous cells.
[0036] In one embodiment, the thickness of mesh graft can range
from 30-300 microns. Preferably, the mesh graft thickness ranges
from 0.5-0.75 mm. A graft of tissue (for example, dermal collagen
plus fibroblasts overlaid with keratinocytes tissue) that is too
thick can result in a too rapid ischemic cell death, especially for
the keratinocyte layer residing above the dermal fibroblast
collagen layer. By contrast, this mesh graft tissue can "take" in
wound sites.
[0037] The epidermal equivalents of the present invention may range
in size from approximately 6 mm to approximately 2.5 cm in
diameter, with a preferred diameter of 2.5 cm. For practical
reasons, the experiments disclosed herein were performed with
epidermal equivalents of approximately 2.5 cm in diameter.
[0038] In one embodiment, the preferred range for epidermal
equivalents is 50-150 microns. In a particular embodiment, the
epidermal equivalents are very thin (thinner than is generally used
in the art, for example, 60 microns). It has been hypothesized that
making the autologous graft too thick will prevent a proper blood
supply from being established, so that the epidermis will not
"take" at the wound site. By contrast, the epidermal equivalents of
the invention can "take" in wound sites.
[0039] In many cases, however, the skin or epidermal equivalents
will have to be delivered from the facility where they are
generated to the institution where they are used. Therefore a
system is needed to enable the transport of the skin or epidermal
equivalents, which have been kept in a condition ready for
grafting. Irrespective of whether the microporous membrane is
removed from the basal cell layer before transport conditions
resembling those during cultivation seem to be favorable. In order
to keep the skin or epidermal equivalents in contact with medium
only from the basal layer, (i.e., during cultivation), agarose in a
concentration ranging from 0.1% to 5%, and preferably in a
concentration of 0.5% to 1%, or methyl cellulose, or any other
gelifying substance in comparable concentrations, may be used to
solidify the transport medium. The skin or epidermal equivalents
will be placed with their basal layer down on the membrane of an
insert previously embedded on top of the solidified or gelled
medium. The multiwell dish containing these inserts is then put in
a blister sealed by a tyvek cover, and shipped. The skin or
epidermal equivalents are, most preferably, used for grafting
within 24 to 48 hours of initial packaging.
[0040] To improve the stability of the epidermal equivalents, the
technique of placing a carrier membrane on top, i.e., onto the
cornified aspect, of the epidermal equivalents and eventually
adhering to it was developed. As an adhesive, fibrin glue is
preferred, however, other options, including, but not limited to:
extracellular matrix components such as collagen, fibronectin,
proteoglycans (e.g., hyaluronic acid, chondroitin sulfate, and the
like), or basement membrane zone components (e.g., laminin,
Matrigel.TM., or L-polylysine), or similar tissue glues, may also
be utilized.
[0041] The carriers utilized in the present invention may consist
of a synthetic membrane, made from at one or more of the following
materials (polyester, PTFE or polyurethane); from one or more
biodegradable polymers (e.g., hyaluronic acid, polylactic acid or
collagen); or a silicone or vaseline gauze dressing, or any other
material suitable for wound dressing. These materials which are
suitable for wound dressing allow the carrier to remain in place to
immobilize the implanted dermal or epidermal equivalents for
several days, rather than requiring the carrier to be removed
immediately after the dermal or epidermal equivalents are
transplanted. Thus, the carrier not only enhances stability and
improves handling, but it also serves as a protective coat against
physical damage as well as the proteolytic milieu and bacteria in
the wound. Moreover, it serves for orientation of the graft (i.e.,
basal side down. cornified side up).
[0042] The skin or epidermal equivalents put onto the carrier have
to be kept in a condition ready for grafting. Irrespective of
whether the microporous membrane is removed from the basal cell
layer for transport conditions resembling those during cultivation
seem to be favorable. In order to keep the skin or epidermal
equivalents in contact with medium only from the basal layer (i.e..
during cultivation), agarose in a concentration ranging from 1% to
5%, and preferably in a concentration of 1 to 3%; methyl cellulose;
or any other gelifying substance in comparable concentrations, may
be used to solidify the medium. The epidermal equivalents together
with the carrier will be placed with their basal layer on top of
the solidified or gelled medium. The whole device is then sealed in
an air tight manner, and shipped. The epidermal equivalents are,
most preferably, used for grafting within 24 hours of initial
packaging.
[0043] The skin or epidermal equivalents are transplanted by simply
placing them in the bed of the wound or other skin defect.
Preferably the skin or epidermal equivalents are then immobilized
(patients are immobilized for 2 hours). The preferred method for
immobilization is by use of a biodegradable material, by some sort
of tissue glue or adequate bandage. As previously described, the
bed of the skin defect can be treated with blood releasates or the
medium from the organotypic culturing prior to, or concomitantly
with, the transplantation.
[0044] In work using encapsulated cells devices (100 micron
membrane, 200-250 microns to the center of the hollow fiber), good
survival of human dermal fibroblasts has been obtained at 300
micron distances from the nearest blood vessel.
EXAMPLE 1
Preparation of ORS Cells
[0045] Keratinocyte precursor cells from the outer root sheath
(ORS) of the hair follicles are selected and subsequently cultured
by use of the following methodology, as disclosed in the present
invention.
[0046] Approximately 40 hair follicles were plucked with tweezers
from the occipital scalp of individuals, and those in the anagen
phase, as detected, for example, by well-developed root sheaths,
were then selected under the dissecting microscope (see e.g. Limat
& Noser, 87 J. Invest. Dermatol. 485-488 (1986); Limat et al.,
92 J. Invest. Dermatol. 758-762 (1989)). The anagen hair was placed
directly on the microporous culture insert without performing the
previously-utilized micro-dissection to remove the hair bulbs and
the infundibular hair shaft.
[0047] Generally, six anagenic hairs were explanted on the
microporous membrane of a cell culture insert (Costar) that carried
on its undersurface a preformed feeder layer preferably comprised
of 20.times.10.sup.3 postmitotic human dermal fibroblasts (HDF) per
cm.sup.2. (see e.g., Limat et al., 92 J. Invest. Dermatol. 758-762
(1989)). The HDFs were derived from skin explants of a healthy,
repeatedly HIV--serology negative and hepatitis-serology negative
individuals and cultured in DMEM supplemented with 10% fetal calf
serum (FCS), or preferably less than 5% human serum, or most
preferably 2% human serum.
[0048] For the purpose of obtaining an efficient outgrowth of the
outer root sheath (ORS) cells from the anagen hair and a high
proliferation rate, it is important not to place the HDF feeder
cells at the bottom of the culture dish, resulting in an additional
medium layer between the HDF layer and the microporous membrane
supporting the ORS cells. Growing each cell type at one side of the
microporous membrane allows a very close interaction, but prevents
cross contamination of the ORS cells with fibroblasts and thus
guarantees a pure culture of ORS cells.
[0049] The culture medium which was utilized consisted of
Dulbecco's modified Eagle's medium/F12 (3:1 v/v) supplemented with
2% human serum, 10 ng of epidermal growth factor per ml of culture
medium, 0.4 microgram of hydrocortisone per ml, 0.135 mM adenine,
and 2 nM triiodothyronine (all obtained from Sigma Chemical Co..
St. Louis, Mo.). The preferred final Ca.sup.2+ concentration of the
culture medium is 1.5 mM (see e.g., Wu et al., 31 Cell 693-703
(1982); Limat & Noser, 87 J. Invest Dermatol. 485-488 (1986)).
Within about 2 weeks, the ORS cells had expanded and reached
confluence. They were dissociated with 0.1% trypsin/0.02% EDTA
mixture, checked for viability, and used for preparation of
epidermal equivalents. It should be noted that, although initial
cultures had been performed using 10% fetal calf serum (FCS;
Boehringer Mannheim, Germany). current utilization of human serum
in order to reduce the number of allogeneic ingredients, provided
superior outgrowth and proliferation of the ORS cells. The human
serum is preferably utilized in a concentration of less than 5%,
and most preferably in a concentration of 2%.
[0050] Explanting plucked anagen hairs directly on the membrane of
culture inserts carrying postmitotic HDF on the undersurface as
feeder cells proved to be a simple efficient and reproducible
method for establishing primary cultures of ORS cells.
Approximately 80% of the explanted hair follicles gave rise to
outgrowth of ORS cells, even when derived from individuals aged
more than 90 years. After 14 days, large areas of the insert were
covered by compactly arranged small cells, at which time they were
used for preparation of epidermal equivalents of the present
invention.
[0051] The comparison of the growth behavior of 70 strains of ORS
cells, which were derived from a total of 30 donors, demonstrated
no significant differences between the young (i.e., 21 donors aged
19-50 years) and the old (i.e., 9 donors aged 51-93 years) donors.
Approximately 5.times.10.sup.5 cells were generally obtained per
explanted follicle and the overall degree of cell viability was
typically higher than 95%. In contrast, in the absence of
postmitotic HDF as a feeder layer, there was only sporadic
outgrowth of ORS cells from the explanted follicles.
EXAMPLE 2
Preparation of Epidermal Equivalents
[0052] ORS cells harvested from primary cultures were seeded at a
density of 30.times.10.sup.3 cells/cm.sup.2 to 100.times.10.sup.3
cells/cm.sup.2, and preferably 60.times.10.sup.3 cells/cm.sup.2, on
cell culture inserts (Costar) which had been previously inoculated
with 10.times.10.sup.3 cells/cm.sup.2 to 50.times.10.sup.3
cells/cm.sup.2, and preferably 20.times.10.sup.3 cells/cm.sup.2, of
postmitotic HDF cells on the undersurface of their microporous
membrane. Similar to the culture of ORS cells, it is important to
keep the HDF feeder cells in close proximity with the ORS cells,
while concomitantly keeping them separated by use of the
microporous membrane. This culture technique enhances
proliferation, differentiation, and thus the homeostasis of the
developing tissue.
[0053] Culture medium was identical that that utilized for the
preparation of the primary cultures as described supra. After 72
hours, the ORS cells were exposed to air by aspiration of the
liquid medium inside the insert (i.e. leaving the underside of the
insert in contact with medium) and cultured for an additional 12-14
days, with three medium changes per week. Alternatively, after one
week lifted culture serum may be totally omitted.
[0054] For transplantation, the so-far-utilized protocol, which is
generally employed for preparation of the fully differentiated
epidermal equivalent for wound grafting, requires the physician to
carefully cut the entire perimeter of the culture insert with a
scalpel blade so as to facilitate the release of the insert
membrane (with undercoated human dermal fibroblasts) with the
attached skin patch squamous-side up. The skin patch is then
released from this membrane by peeling with a fine forceps and
placed, basal-side up, on a new membrane disk in a culture dish for
eventual transplant to the patient. This aforementioned procedure
is both laborious and time consuming, and can lead to reversal of
the basal and squamous orientation.
[0055] A markedly simpler method which utilizes a carrier membrane
patch cap has been devised which utilizes a membrane patch cap
(analogous to the fibrin glue patch procedure described below)
which is placed directly on top of the squamous surface layer. The
membrane cap can then be easily grasped together with the skin
patch below using fine forceps and peeled from the culture insert
well surface, and. e.g., after incubation in diluted Dispase
solution, be peeled from the culture insert membrane. The membrane
can then serve a plate for placing the graft onto the wound without
mixing up the orientation of the graft (i.e., basal side down,
squamous side up).
[0056] For stabilization and as a protective coating in case of
grafting, the epidermal equivalents of the present invention are
coated on top with diluted fibrin glue, which also serves to
clearly identify the upper (i.e., cornified) side. Fibrin glue, the
preferred embodiment of the present invention, is a generally
accepted, natural human product which is used extensively as a
tissue glue. By applying a thin coating of fibrin glue (which is
clearly visible with the naked eye) to the cornified squamous
air-exposed surface of the epidermal equivalent, the physician
placing the epidermal equivalent onto the wound site will be fully
assured of proper graft orientation (i.e., the basal surface of the
skin patch will always be the side that does not have the clearly
visible fibrin glue cap). Previously, in many instances, during the
preparation of the epidermal patch for wound grafting, the
orientation of the patch becomes confused. Should the skin patch be
placed in squamous-side down orientation onto the graft site, there
would be significantly decreased likelihood of a successful graft.
Thus, the use of this simple "marking" completely eliminates this
problem.
[0057] In addition, anti-microbial and/or anti-fungal substances
may also be included in the fibrin glue, so as to impede any
possible microbial contamination or overgrowth of the graft. Many
chronic lesions are chronically-infected, which can result in the
inhibition of graft "take" and subsequent wound healing following
the initial skin grafting. Moreover, the addition of one or more
antibiotics or anti-fungal agents by direct emulsification within
the fibrin glue surface cap, may provide a significant improvement
in the delivery of sufficient quantities of anti-microbial agents
to the transplant site.
[0058] It should be noted that the ORS cells which were harvested
from primary cultures, and cultured at the air-liquid interface on
insert membranes carrying postmitotic HDF at their undersurface,
typically developed a stratified epithelium within 14 days. This
stratified epithelium consisted of a basal layer of small cuboidal
cells below a thick suprabasal compartment of progressively
flattened cells. A prominent granular layer, as well as an
orthokeratotic horny layer were also found to be present.
[0059] Based upon the experimental finding of approximately 80% of
the follicles giving rise to ORS cell outgrowth, approximately five
anagen hair follicles were required for the generation of 1
cm.sup.2 of epidermal equivalents. The period to generate graftable
epidermal equivalents usually was four weeks in toto (i.e., two
weeks for the primary culture and two weeks for the subsequent
organotypic culture).
EXAMPLE 3
Stabilization
[0060] Before delivery, the epidermal equivalents are "coated
on-top" by placing a silicone membrane of an appropriate diameter
onto the cornified upper aspect of the cultures. To further enhance
stability, e.g., in case of thin and/or large epidermal
equivalents, as well as to increase adhesion of the silicone
membrane, a thin layer of tissue glue, e.g. fibrin glue, may be
applied before.
[0061] On-top coating (1) enhances stability and improves handling
of the grafts and (2) serves as a protective coat against physical
damage as well as the proteolytic milieu and bacteria in the
wound.
EXAMPLE 4
Shipping
[0062] On-top coated epidermal equivalents are detached from the
culture insert membranes by incubation in diluted Dispase and then
grasping the epidermal equivalents together with the silicone
membrane using fine tweezers and transferring them on the membrane
of an insert previously embedded in 0.7% agarose soaked with
culture medium in the well of a multiwell dish. These dishes are
then placed in the shipping container. For application to the wound
bed, the epidermal equivalents are again grasped, together with the
silicone membrane, which (1) serves for orientation of the graft
(i.e., basal side down, cornified side up) and (2) by leaving it on
the grafted epidermal equivalents in the wound serves as a
protective coat (see above).
EXAMPLE 5
Successful Treatment of Chronic Leg Ulcers with Epidermal
Equivalents Generated from Cultured Autologous Outer Root Sheath
Cells
[0063] The outer root sheath cells of hair follicles can substitute
for interfollicular epidermal keratinocytes, as during healing of
skin wounds when these cells migrate onto the denuded area and
contribute to epidermal regeneration (Limat et al, 107(1) J.
Invest. Dermatol. 128-35 (1996), incorporated by reference). Using
the improved culture techniques of the invention, we generated
epidermal equivalents from cultured outer root sheath cells of
patients suffering from recalcitrant chronic leg ulcers, primarily
of vascular origin. In such epidermal equivalents, tissue
organization as well as immunolocalization of epidermal
differentiation products (keratin 10, involucrin, filaggrin) and
integrins were indistinguishable from normal epidermis. As
determined by the number of bromodeoxyuridine-incorporating cells,
the basal layer contained a large compartment of proliferative
cells irrespective of donor age. FACS analysis of the outer root
sheath cells, used to prepare the epidermal equivalents, disclosed
a fraction of small cells with enhanced expression of
.beta.1-integrin, a potential stem cell marker, in contrast to
acute wounds, a major definitive take of grafted cultured
autologous keratinocytes has not been convincingly demonstrated in
chronic wounds. Grafting of epidermal equivalents generated in
vitro from autologous outer root sheath cells on 11 ulcers in five
patients resulted in a definitive take rate of about 80%, with
subsequent complete healing within 2 to 3 weeks of five out of
seven ulcers grafted with densely arranged cultures. This
improvement in the treatment of chronic leg ulcers with cultured
autologous keratinocytes probably depends on the large compartment
of proliferative cells as well as on a well-developed horny layer
which prevents disintegration of the grafts. Practical advantages
of the new technique are its noninvasiveness, the lack of need for
surgical facilities or anesthesia, and a short immobilization
period after grafting.
[0064] In Vitro Experiments. Cell Cultures. About 40 hair follicles
were plucked from the occipital scalp of individuals aged up to 91
years , and those in the anagen phase selected under the dissecting
microscope. The hair bulbs as well as the infundibular parts were
removed with microsurgical blades. Usually, six follicles were
explanted on the microporous membrane of a cell culture insert
(Falcon 3090; Becton Dickinson, Franklin Lanes, N.J.) that carried
on its undersurface a performed feeder layer made of 10.sup.5
postmitotic human dermal fibroblasts. Culture medium consisted of
Dulbecco's modified Eagle's medium/F12 (3:1) supplemented with 10%
fetal calf serum (Boehringer Mannheim, German), 10 ng of epidermal
growth factor per ml, 0.4 Fg of hydrocortisone per ml, 0.1 nM
choleratoxin, 0.135 mM adenine, and 2 nM triiodothyronine (all from
Sigma Chemical Co., St. Louis, Mo.), final Ca.sup.2+ concentration
1.5 mM. Within about 2 wk the ORS cells expanded and reached
confluence. They were dissociated with trypsin/EDTA 0.1%/0.02%,
checked for viability, and grown either in secondary cultures in
keratinocyte growth medium (KGM containing 0.15 mM Ca.sup.2+;
PromoCell, Heidelberg, Germany) or used for flow cytometry analysis
and preparation of epidermal equivalents (see, below). For
long-term storage in liquid nitrogen, they were frozen in KGM
containing 10% fetal calf serum and 10% dimethylsulfoxide.
[0065] For comparison, primary cultures of ORS cells were also
established by trypsinization of hair follicles and plating the
disaggregated ORS cells on a preformed feeder layer made of
postmitotic fibroblasts, as previously described (Limat et al,
1989). To avoid confusion, follicles obtained by this method are
referred to as "trypsin-treated follicles."
[0066] Fibroblasts were derived from skin explants of a healthy,
HIV-serology, and hepatitis-serology-negative individual and
cultured in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum.
[0067] Flour Cytometry. The following mouse monoclonal antibodies
(mAbs) of 1 gG.sub.1 subtype reacting with different integrin
chains were used: 4B4 with the .beta..sub.1-chain (Coulter,
Hialeah, Fla.), 5E8 with the .alpha..sub.2-chain, J143 with the
.alpha..sub.3-chain, Lv 230 with the .alpha..sub.v-chain, and MT78
with the .alpha..sub.6-chain. MAb 439-9B recognizes the
.beta..sub.4-chain.
[0068] ORS cells at 1.times.10.sup.6/ml were washed once with
phosphate-buffered saline, 1% fetal calf serum, and 0.02% NaN.sub.3
at 4.degree. C. and reconstituted with 1 ml of the same buffer. A
100 .mu.l cell suspension was then incubated with 0.1 .mu.g of mAbs
or isotype control antibody (Dako, Glostrup, Denmark) for 25 min at
4.degree. C. After being washed twice with the same buffer, cells
were incubated with a phycoerythrin-labeled polyclonal goat
anti-mouse anti-body (Dako) for another 25 min at 4.degree. C.,
washed again, and subsequently fixed with the above-mentioned
buffer supplemented with 2% paraformaldehyde. Cells were analyzed
on a 4-logarithmic scale EPICS Profile II flow cytometer equipped
with a power pack, and data were analyzed using the ELITE software
(Coulter).
[0069] Epidermal Equivalents. ORS cells harvested from primary
cultures were seeded at a density of 5.times.10.sup.5/cm.sup.2 on
cell culture inserts (Falcon 3095) carrying 5.times.10.sup.4
postmitotic fibroblasts on the undersurface of their microporous
membrane. Culture medium was the same as for the preparation of
primary cultures. After 24 hr. the ORS cells were exposed to air by
aspiration of the medium inside the insert and then cultured for 12
to 14 days with three medium changes per week. In some cultures, 65
.mu.M 5-bromo-2'-deoxyuridine (BrdU; Sigma) were added for the
final 18 hr.
[0070] For histologic analysis, the epidermal equivalents were
excised from the insert with a 6 mm punch (Stiefel Laboratorium,
Offenbach am Main, Germany), fixed in 5% formalin, and processed
further together with the underlying insert membrane according to
standard procedures. For immunohistologic examination, the
epidermal equivalents were similarly punched out, but then
separated from the insert membrane by fine tweezers, snap-frozen in
liquid nitrogen-cooled isopentane, and stored at -80.degree. C.
until processing.
[0071] For indirect inmnunofluorescence, cryostat sections of 6
.mu.m were air-dried. fixed with ice-cooled acetone/ethanol (1:1),
rehydrated with phosphate-buffered saline, blocked for 15 min with
nonimmune serum, and incubated at room temperature for 60 min with
the primary antibodies and, after extensive washing, for 45 min
with the secondary antibodies. The following mAbs were used as
primary antibodies: Ks 8.60, mainly reacting with keratin (K) 10
and weakly with K1, diluted 1:20 (Sigma); anti-human involucrin,
diluted 1:100 (Sigma); anti-human profilaggrin/filaggrin, diluted
1:100 (BTI, Stoughton, Mass.); 4B4 directed against the
.beta..sub.1-integrin chain, diluted 1:10 (Coulter). Secondary mAbs
against mouse IgG conjugated with fluorescein isothiocyanate were
purchased from Sigma. As negative controls, sections were incubated
with non-immune serum and conjugated secondary antibodies, which
revealed in a few cases weak diffuse staining of fully keratinized
areas.
[0072] For the determination of BrdU-positive cells, cryostat
sections were denatured in 1.5 M HCl and successively incubated
with 0.5 .mu.g/ml Hoechst 33258 for 30 min, mAb anti-BrdU (Partec,
Arlesheim, Switzerland) diluted 1:100 for 45 min, and fluorescein
isothiocyanate-linked anti-mouse IgG (Sigma) diluted 1:30 for 45
min. The percentage of BrdU-positive cells in the basal layer was
determined in epidermal equivalents prepared from ORS cells of two
leg ulcer patients aged 72 and 91 years (n=4; two epidermal
equivalents per patient). For each epidermal equivalent, about 2500
basally located nuclei in 10 randomly selected sections were
counted.
[0073] For transplantation, the epidermal equivalents were excised
from the insert together with the underlying membrane using a 6-mm
punch (Stiefel Laboratorium) and positioned upside-down on a
punched-out polyester membrane (Thomapor 95877; Reichelt Chemie,
Heidelberg, Germany) of 6 mm diameter. In one patient additional
epidermal equivalents of 8 mm diameter were prepared likewise. The
insert membrane together with the attached postmitotic fibroblasts
was then carefully removed with fine tweezers. The epidermal
equivalents on their supporting polyester membrane were washed in
Dulbecco's phosphate-buffered saline and left floating therein
until their application on the wound bed, usually for no longer
than 30 min.
[0074] Autologous Grafting in Chronic Leg Ulcers. With the approval
of the Ethics Committee of the University of Berne and after
obtaining written informed consent, five in-patient (one male, four
females, aged 58 to 91) suffering from recalcitrant chronic leg
ulcers (four of them with more than two ulcers on the same leg,
duration at least 4 years; venous or mixed arterial and venous
disease in four, in one additional diabetes mellitus, primary
lymphoedema in one) were enrolled in a pilot study. The ulcers were
cleaned conventionally (primarily with hydrocolloidal dressings and
topical antimicrobial agents) until ready for grafting. Then up to
20 autologous epidermal equivalents, usually 6 mm, in one ulcer 8
mm in width, were placed, basal layer downward on the surface of
the ulcers, and the supporting polyester membranes were carefully
removed with fine tweezers. This grafting procedure was performed
at the bedside; no anesthesia was needed. In four of the patients,
further ulcers on the same leg served as controls. All ulcers were
then covered with a transparent, semiocclusive dressing (Tegaderm;
3M, London, Canada) overlaid by an elastic bandage with compression
adapted to the patient's arterial status. The patients were
immobilized for 2 h immediately after grafting. After 3 d, the
semiocclusive dressing was carefully removed and a hydropolymer
dressing (Tielle: Johnson & Johnson Medical, Ascot,. UK)
applied, again overlaid by the elastic bandage. The hydropolymer
dressings were then changed every 2 to 5 days. After complete
re-epithelialization local treatment was switched to topical
emollients, and the patients were instructed to adhere to a
long-term compression therapy adapted to their arterial status.
Take of the grafts and healing of the ulcers was documented by
standardized photographs taken on each change of the dressings.
[0075] In Vitro ORS Cells Differentiate Into Epidermal Equivalents
Similar to Normal Epidermis. Explanting plucked anagen hair
follicles directly on the membrane of culture inserts carrying
postmitotic fibroblasts as feeder cells at their undersurface
proved to be a simple, efficient, and reproducible tool for
establishing primary cultures of ORS cells. About 80% of the
explanted hair follicles gave rise to outgrowth of ORS cells, even
when derived from individuals aged up to 91 years. After 14 days,
large areas of the insert were covered by compactly arranged small
cells, at which time they were used for the preparation of the
epidermal equivalents. In contrast, ORS cells derived from the
trypsin-treated follicles exhibited a less compact arrangement with
numerous cells of a larger size. Comparison of the growth behavior
of 70 strains of ORS cells derived from 30 donors revealed no
significant differences between young (21 donors aged 19 through 50
years) and old donors (9 donors aged 51 through 93 years), since
about 0.5.times.10.sup.6 cells were usually obtained per explanted
follicle. Cell viability was higher than 95%. In the absence of
postmitotic fibroblasts, there were only sporadic outgrowth of ORS
cells.
[0076] Because a logarithmic linear relationship between the
relative level of .beta..sub.1-integrin on the cell surface and the
proliferative capacity of keratinocytes has been postulated, we
compared the expression of integrins in primary cultures of ORS
cells established by the two different techniques, i.e., ORS cells
from explanted follicles or from trypsin-treated follicles. ORS
cells from four different donors grown by both techniques were
analyzed by flow cytometry. On the basis of their light-scattering
characteristics, the cells could be subdivided into two groups;
group A, with a distinctly lower forward light scatter, i.e.,
smaller cell size, and group B, with higher forward light scatter,.
thus having a larger cell size. For ORS cells derived from
explanted follicles, group A accounted for about 4% and group B for
72% of the total cell number, while values of 2.6% and 75%,
respectively, were found for ORS cells grown from trypsin-treated
follicles (mean values of four separate experiments). In group A,
the percentage of cells staining for
.beta..sub.1-.beta..sub.4-integrins as well as the mean
fluorescence per cell of the .beta..sub.1-and to a lesser extent
also the .alpha..sub.2-, .alpha..sub.3-, .alpha..sub.v-integrins,
were higher in ORS cells grown from explanted follicles than in
those from trypsin-treated follicles. In group B, no differences
were detected in the two culture techniques, neither in the
percentage of integrin-positive cells nor in the mean fluorescence
per cell.
[0077] ORS cells harvested from primary cultures and plated on
insert membranes carrying postmitotic fibroblasts at their
undersurface developed a stratified epithelium within 14 days. This
consisted of a basal layer of small cuboidal cells below a thick
suprabasal compartment of progressively flattened cells. A granular
layer and a mostly orthokeratotic horny layer were present.
[0078] The immunolocalization of epidermal differentiation products
was identical to that found in normal epidermis. Thus, the
differentiation-specific K10 was absent in the basal layer, but
strongly expressed suprabasally from the second layer on.
Involucrin displayed its typical honey-comb pattern form the
mid-stratum spinosum on, whereas the granular staining of filaggrin
formed a continuous band beneath the horny layer. As in normal
epidermis, the reactivity of the .alpha..sub.2-, .alpha..sub.3- and
.beta..sub.1-chains of integrins was distributed over all aspects
of the plasma membrane of the basal cells, displaying decreasing
intensity with progressive differentiation.
[0079] BrdU-positive cells were found predominantly in the basal
layer of the epidermal equivalents and accounted for 24% of the
basal cells [597.+-.21 BrdU-positive cells for 2464.+-.115 basal
cells (mean.+-.SD): n=4].
[0080] Based on 80% of follicles giving rise to ORS cell outgrowth,
about five anagen hair follicles were needed to generate 1 cm.sup.2
of epidermal equivalents. The period to generate graftable
epidermal equivalents usually was 4 weeks i.e., 2 weeks for the
primary culture and 2 weeks for the organotypic culture.
[0081] Autologous Epidermal Equivalents Are Grafted Successfully on
Chronic Leg Ulcers. A total of 11 ulcers were treated, seven of
them by covering about 90% of the ulcer surface with densely
arranged cultures, four by putting isolated cultures into the
central parts. On the first change of the dressing 3 d after
grafting, about 80% of the grafts were visible and adherent to the
wound bed in both types of treatment. Within the following 2 to 3
wk the grafts consolidated in five of the seven densely grafted
ulcers, resulting in complete re-epithelialization and healing. In
the two remaining, chronically infected (Pseudomonas) ulcers, the
grafts were partly destroyed, which led to delayed healing by 4 to
5 weeks. In the ulcers treated by isolated grafts, there was
accelerated formation of granulation tissue and
re-epithelialization mainly from the wound edges, as compared to
the ulcers on the same leg treated with the dressings only. In this
type of treatment, permanent take with subsequent expansion of the
grafts resulting in complete re-epithelialization was only
documented for one ulcer treated with larger epithelial sheets
measuring 8 mm in diameter. The control ulcers in the four patients
with more than two ulcers on the same leg were only slightly
improved after 3 weeks, at which time they were treated either by
further grafting of autologous epidermal equivalents or by
conventional surgery.
[0082] After re-epithelialization, the epidermis was initially
still fragile with some tendency to blistering after minor
frictional trauma, occasionally resulting in small erosions. These
erosions re-epithelialized rapidly under conventional topical
treatment. The first patients have now been followed up for 6 mo
and show increasing stabilization of the treated areas and no
recurrence of the ulcers so far.
[0083] From the foregoing detailed description of the specific
embodiments of the present invention, it should be readily apparent
that a unique methodology for the selection and culture of
keratinocytes from the outer root sheath (ORS) of hair follicles
for subsequent use in, for example, skin grafting procedures, has
been described. Although particular embodiments have been disclosed
herein in detail, this has been done by way of example for purposes
of illustration only, and is not intended to be limiting with
respect to the scope of the appended claims which follow. In
particular, it is contemplated by the inventor that various
substitutions, alterations, and modifications may be made to the
invention without departing from the spirit and scope of the
invention as defined by the claims. For example, the selection of
anagen hairs are believed to be a matter of routine for a person of
ordinary skill in the art with knowledge of the embodiments
described herein.
* * * * *