CA2230263A1 - Reconstituted skin - Google Patents

Abstract

The invention of this disclosure relates to a method for producing composite skin comprising a dermis inoculated directly with mammalian cells. In the method of this invention a dermis is obtained, preferably an acellular dermis of xenogeneic or human origin. The cells used to inoculate the dermis are transferred directly or preselected for properties consistent with progenitor or stem cell. The cells are of xenogeneic or human origin. In the practice of the preferred embodiments of this invention cells inoculated on the dermis are allowed to propagate ex vivo in culture to increase their number. In the preferred embodiment of this invention the ex vivo culturing results in the expansion of a population of cells having progenitor characteristics. The cells used to inoculate the dermis, in the preferred embodiments of this invention are genetically modified ex vivo. In the most preferred method of this invention the dermis to be inoculated is of human origin. In this embodiment the cells are either autologous or allogeneic in origin or a combination of both. The inoculated human dermis of this preferred embodiment can be applied to a human or utilized ex vivo as a laboratory assay.

Description

RECONSTITUTED ~KIN
. 5 This invention relates to methods for the development of a reconetit~lte~l skin composite for transplantation. These methods will lead to the production a reconetit~lte~l skin consisting of an intact, biological, acellular, dermal matrix in combination with epidermal cells which can serve as a repl~ em~nt for full-thickness 10 skin defects.

Currently, the standard grafting procedure for full thickness skin injury involves the use of autologous split thickness skin grafts (STSG). While this has been shown to be a life saving procedure~ there is still a need for improvement with regard to donor site trauma and the final cosmetic and functional outcome of the original wound. In full-thickn~e~e skin injuries, STSG must pro~ide both dermal and epidermal components at the wound site. When autologous donor sites are limit~fl, the STSGmust be meshed and e~cp~n~lecl to allow coverage of the entire wound area. This meehP~l configuration leaves areas of the wound uncovered by both dermis and epidermic The epithelial cells ofthe grafted epidermis will eventually migrate into and cover the interstices of this mesh pattern and thereby promote wound closure.
However, as dermis is not a regenerative tissue, problerns with scarring and contracture arise later when the grafts contract due in part to a lack of sufficient dermis.
Another approach when STSG donor sites are limited is to culture epithelial cells from a biopsy of fresh healthy skin. These cells c~n be e~cp~n-le-l in culture to allow coverage of a much larger area than the original biopsy site. Research to investigate the conditions nçcese~ry to grow keratinocytes in vitro began to emerge in the late 1960's and early 1970's. This research was aimed at defining the culture 3 o conditions n~cçes~ry to propagate non-transformed keratinocytes for extended periods in vitro. Pioneering studies in this area were performed by Rheinwald and Green.One of the most important fint1in~e in these early investigations was the use of mouse W O 97/08295 -2- PCTnJS96/13616 fibroblast 3T3 feeder layers and culture medium supplemented with epidermal growth factor (EGF).EGF was found to be a powerful mitogen for keratinocyte growth and allowed these cells to be prop~g~ted for several passages in vitro. Several other additives including cholera toxin, insulin and insulin-like growth factor, bovine piLuiL~y extract, hydrocortisone and fetal bovine serum, to name a few, have since been found to be important additives for keratinocyte culture. This l~se~cll led to the development of a serum-free, fully defined medium (MCDB-153) developed by Boyce and Ham. Together, these fintlings have allowed researchers to propagate keratinocytes in vitro for 10+ passages.
o The culture of epithelial cells from a skin biopsy involves the separation of the epidermis from the dermis, followed by dissociation of the cells present in the epidçrmi~ This can be accomplished using one or a combination of the following enzymes and chemicals to separate the two different layers of the skin: Dispase,Thermolysin, trypsin, or ethylçn~ min~tetraacetic acid (EDTA). The separated epidermis is then inrllh~tçcl further in trypsin plus EDTA to dissociate the epidermis into a cell suspension. ~ It~rn~tively, it has been shown that microdissection of hair follicles followed by trypsin/EDTA incubation will also led to the growth of a population of keratinocytes. The dissociated cells are then placed into culture medium with a combination of growth factors, presence or ~hS~?n~e of serum, and presence or absence of irradiated or mitomycin treated mouse fibroblast. If the cells are then allowed to remain in culture to or excee~ing confluence, they will form an intact sheet of keratinocytes. This sheet can then be released from the culture vessel by treating with enzymes such as Dispase which disrupt the ~tt~ ment of cells to the ~ub~LIdt~ but do not disturb cell-cell contacts.
These intact sheets of autologous keratinocytes (referred to as cultured epithelial autografts or CEA) can be produced from a small biopsy obtained from the patient. The production of these sheets however requires weeks of culture time.
Although initial interest and use of CEA technology was high, as long term results became available it was evident that the lack of dermal repl~cement imparts significant limitations on this approach including low overall take rates, sc~rrin~, and imm~tnre b~c~ment membrane formation leading to fragility of the epi~lçrmic..
An ~ liti~n~l ~ltern~tive for covering extensive burn wounds is microm~hing 5 or microskin grafting. When autologous donor sites are limited, the available STSG
can be meshçcl and widely exr~n~le~l (generally at a ratio of 4:1 or greater) or minced by passing the tissue multiple times in dirr~,.cnt orientation through a standard mesher.
While studies have shown that widely me~hP~ autografts can eventually close a large full-thickness skin wound, these grafts a) take a long time to re-epithelialize 10 interstices of the meshed graft, b) result in a "cobblestone" a~e~dl~ce at the graft site and c) often lead to debilit~tin~ scarring and contracture. As is the case for CEA
grafting, the lack of dermal repl~ " in these procedures presents significant limitations on the final cosmetic and functional outcome. The use of an acellular dermal matrix, with an intact b~Pment membrane complex, in combination with thistechnique will allow for a better cosmetic and functional outcome at the wound site.
Epithelial cells which migr~te from the microskin pieces onto the b~PmPnt membrane of the acellular dermis will also eventually migrate under the microskin pieces causing them to be sloughed from the snrf~ce. This will result in a smoother graft surface devoid of the cobblestone appearance. The ~l~sence of the acellular dermal matrix will also decrease scarring and contracture at the graft site.
In order to be fully effective, a graft for full thicknPcs burn wounds should have the following characteristics: a) replace both lost dermis and epi~ermi~, b) not require extensive in vitro cell culture to produce the graft, c) deliver a persi~tpllt dermis and epidermis, and d) require only one surgery and thereby reduce patientmorbidity and mortality and reduce costs as a result of shorter hospital stays.
The invention of this patent includes the use of an intact acellular dermal matrix in combination with epithelial cells to reconstitute a composite skin meeting these requirements.

W O 97/08295 -4- PCT~US96/13616 Dermal Matrices and In Vitro Recon~lilul~d Skin: One technique for producing reconstituted skin involves using deepid~rmi7~l dermis (DED), which was first investig~t~d by Prunieras et al.. This dermal matrix is generally produced by 5 prolonged incubation ( > 4 weeks ) of hDan skin in phosphate buffered saline or repeated freezing and thawing of the skin which kills all of the cells of the dermis and epi~lermic Results with this technique have been variable. Human trials have demollslldL~d poor take rates in skin wounds using this substrate.
Krejci et al. have e~minçd acellular versus cellular human dermal substrates 10 in the presence or absence of an intact b~cement membrane complex in vitro. They found that papillary dermis lacking fibroblasts but m~ g an intact b~cem~nt membrane, and reticular dermis which had been repopulated with dermal fibroblasts were both good substrates for keratinocyte growth. These results indicate the importance of b~cement membrane and/or dermal fibroblasts for the production of an l S in vitro skin.
As stated above, there have been nDerous studies which report the nececcity of fibroblasts to support a fully differ~nti~t~d epidermis. To date, the majority of derrnal substrates have been composed of animal collagen gels in which hDan fibroblasts are seeded prior to keratinocyte culture. Upon addition of fibroblasts, the collagen matrix contracts to approximately two thirds its original size. Of particular interest regarding these interactions is the report by Krejci et al.. which demon~Lldled that an acellular dermal substrate with the b~c~ment membrane intact was as effective as fibroblast repopulated reticular dermis in supporting recon~iLuLion of an epi~l~rmic An argument can be made that the b~ ment membrane acts as a sink for growth 2s factors, calcium and other as yet llnicl~?ntified substances which are secreted by fibroblasts and keratinocytes. In this way, an optimally preserved acellular dermis with the bS~cement membrane intact can support keratinocyte growth for some period of time. After these stores are depleted, fibroblasts may become nPcess~ry to recoll~LiLuLe these growth factors and m~int~in the epicl~rrni~ Con~i~tent with this hypothesis is a report by Higounenc et al., which found that epidermis reconstructed on DED, while exhibiting morphological and biochemical characteristics very similar to in vivo skin, did not fully norrnalize until after grafting onto an animal.
Attempts have been made to produce a biosynthetic composite skin as reported by Bell et al.. This composite was composed of fibroblasts seeded into a lattice of bovine collagen which was allowed to contract prior to overlying with keratinocytes.
These studies reported the production of a multilayerecl epidermis and the formation of some b~cPment membrane components within 2 weeks. This work was the 10 foundation for the production of CrdrL~killTM (Organogenesis Inc., Canton, MA).
GlcLrLshill is a reconstituted colll~o~ile skin currently in clinical trials for use in chronic ulcer tr~tmPnt In 1988 Boyce, et al. introduced a synthetic, biological dermal substrate similar to that developed by Bell. This substrate was composed of human fibroblasts 5 in a component collagen-glycosaminoglycan matrix. After seeding this substrate with keratinocytes they noted increased production of l~minin and Type IV collagen invitro, and a thicker epidermis with increased cell layers. This composite was then co.,lp~d to cultured epithelial autograft sheets (CEA) for covering full thickness wounds on nude mice. The composite grafts showed increased epidermal rete ridge 2 o formation and better adherence and long term maintenance than the CEA. Reports of human clinical application of this matrix however are l~cking Hansbrough et al. reported on what was described as an improved composite graft over that previously described. This skin substitute consisted of human fibroblasts cultured into a synthetic polyglactin mesh. The composite was further 2S developed at Advanced Tissue Sciences, La Jolla CA., and is now called DermagraftTM. The reported advantage of this substrate was that it could be absorbed by hydrolytic rather than proteolytic degradation. However, results in nude mouse studies failed to show enh~n~ed healing characteristics over those mentioned W O 97/08295 -6- PCTrUS96/13616 previously. Further, despite clinical application there are no reports of sl-ccçc~ful reconstitution of both dermis and epidermis in full-thickness burn injuries.

~4cellr~lnr Dermis: The inventors have previously been granted a patent regarding the procç~in~ and production of an intact, acellular dermal matrix of hurnan or porcine origin (AlloDerm~ and XenoDermTM respectively).
The processing and preservation method was tlecigne~ to generate a transplantable biological tissue graft that specifically meets the following criteria:
(a) provides an extracellular protein and collagen matrix which can be remodeled and repaired by the host, (b) provides an intact b~m~-nt membrane for secure re~tt~chment of viable endothelial or epithelial cells, (c) does not elicit a specific immllne lc~ollse by the host, (d) does not calcify, and (e) can be easily stored and transported at ambient temperatures.
The dermal matrix processed in this manner has been shown to possess all of the major components of the b~cem~ont membrane complex including collagens Type IV
and VII and l~minin Further, the matrix has been shown to be effective as a graft for severe burn wounds by replacing lost dermis, allowing immP~ te infiltration of host fibroblasts and endothelial cells and allowing the use of a thinner autologous split-thickness skin graft (STSG) [as a source for keratinocytes] reSlllting in less trauma to the donor site.

Epidermis: The epidermis is a continll~lly renewing tissue composed primarily ofkeratinocytes. As such, there are at least three functionally distinct types of ker~tinocytes in the epidermis: 1. stem cells (progenitors), 2. transient-amplifying cells (exhibit rapid proliferative growth but for only a limited time), and 3. post-mitotic cells (mature dir~~ ted). In this scheme the stem cell is llltim~tely W O 97/08295 -7- PCTrUS96/13616 res~oll~ible for all keratinocyte replacement in the epi/~çnni~, and therefore is essenti~l for long term m~ te~ re of the organ. Therefore, the ~epidermal stem cell is n~cç~s~. y for the long term pçrci~terlce of a grafted epithelium.
The current literature regarding keratinocyte research contains several references to epidermal stem cells (as detailed below). Unfortunately, unlike the hematopoietic system, specific m~rker~ for the putative epidermal stem cell have not yet been ic1~ntifie-1 The epidermal stem cell has however been associated with several distinct physical and functional characteristics which set it apart from the other ker~tinocytes of the epicltormi~ These ~lv~ ies include: long cell cycle time;
10 enh~nee~l e~ ssion of int~rin~ or other markers including specific cytokeratins;
small cell size relative to other ker~tinocytes; and rapid ~tt~ ment to b~cemt?nt membrane components. These pL'Op.,. Lies can be used to develop a protocol for the isolation and enrichment of epi~1enn~l stem cells for producing a composite skin in combination with an intact ~ce~ r dermal matrix. The isolation of the epiderrnalstem cell is analogous to the methodology used for protein purification where its physical prop~-Lies are known but the sequence of the protein is unavailable. Hence, instead of separation and isolation by molecular weight, charge, solubility or selective adsorption (as used for protein purification) we can selectively enrich for epi~lerm~l stem cells by taking advantage of differences in cell cycle time, cell size, inle~5lill or cytokeratin e~ ssion and ~tt~rhment criteria. The epidermal stem cells can be selectively tagged by taking advantage of their slow cycling time, followed by selective isolation of the tagged cells by differences in size, marker ~ules~ion and/or selective ~tt~ehmPnt to di~le.ll substrates. By taking advantage of the physical and functional p,~ .Lies ascribed to the putative epidermal stem cell we can enrich for these cells and thereby enh~nee the formation of a neoepidermis in a composite graft.

Stem Cell Enrichment bv Attachment: Epit1~rm~l k~r~tiTlocytes which attach most rapidly to b~Pment membrane components have been shown to possess the highest W O 97/08295 -8- PCT~US96/13616 colony forming efficiency. Specifically, research reported by Jones et al. has shown that keratinocytes which attach to collagen type IV coated dishes in as little as 5 minl~t~S have a higher colony forming efficiency than those which take longer toattach. The epidermal progenitor cells may be isolated by performing panning techniques using culture vessels coated with type IV collagen, fibronectin, l~minin, or a combination of these coatings. ~It~rn~tively, p~nning techniques can be ~rc --.-ed using the acellular dermal matrix which has an intact b~cemPnt membrane cont~ining l~minin and collagens type IV and VII, in the correct three ~lim~ncional configuration.
~ltern~tively, partial degradation of the b~crment membrane complex present on the o acellular dermal matrix may be n~cess~ry to mimic a wounded scenario and henceactivate keratinocyte proliferation on the matrix. The b~cem~nt membrane can be partially degraded by enzymatic treatment with Dispase II or Thermolysin. Close attention must be given to the collagen present in the dermis to ensure that thei..le~,.ily of the dermis is not con.plo...ised during these enzymatic tre~tnnrntc.
Keratinocytes can then be seeded onto an area of the treated dermal matrix.

Selective isolation o f the epidermal stem cells bv cell size: In order to take advantage of this ~ropel Iy of epidermal stem cells, keratinocytes can be se~ d by size using either density gradient centrifugation, unit gravity se~liment~tiQn. or sorted by size using a cell sorter.
Density gradient centrifugation has been accomplished with keratinocytes using a continuous colloidal silica (Percoll) density gradient. Using this technique it is reported that 3 fractions of ker~tinocytes can be isolated. This corresponds well with the three proposed types of keratinocytes present in the epidermis (stem, transient-amplifying and termins~lly differenti~t~cl). Alternatively, unit gravity se-liment~tion can be performed. This procedure has been used in different laboratories to separate proliferative and termin~lly differenti~ting subpopulations of keratinocytes. Freshly isolated keratinocytes are placed in a modified sedimentation W O 97/08295 -9- PCT~US96/13616 chamber, from which aliquots of cells are removed and e~minec~ for label retention as defined in the localization studies. The cells can be evaluated for a progenitor phenotype by colony forming efficiency assays (CFE) and growth in soft agar as an index for stem cell isolation. Some potential problems with these techniques include:
5 a) the disaggregation to a single cell suspension must be very efficient to avoid cell clumps which would se~1iment at different rates, and b) the size differential between stem cells and transient-amplifying cells may be as small as 1-2 micrometers making effective segregation very difficult. If the epidermis is not efficiently disag~ gal~d to single cells, clumps of cells may be filtered through sterile cotton or nylon mesh.
10 Although the different keratinocyte subpopulations may be very close in size, these techniques provide some enrichment over non-selected populations.

Stem Cell Enrichment bY~nti-proliferation: Another cell selection technique involves selective killing of rapidly dividing cells (a negative selection process). 5-Fluorouracil, an antirnetabolite, has been used in other systems to kill rapidly dividing or metabolically active cells. In this system, S-FU can be used during in vitro culture conditions to selectively kill transient-amplifying (rapidly dividing) cells which have a short cell cycle time, while sparing the epidermal stem cells which have a longer cell cycle. This can be accomplished by pulse dosing of S-FU during culture of rapidly 20 çxp~n~ling keratinocytes. These conditions may include cl~ltllrin~ in the presence of 1 X 10-7 M retinoic acid for 24 hours followed by addition of 1 X 10-5 M isoproterenol for an additional 24 hours.
Hyperthermic tr.o~trnent of the skin (or keratinocytes in culture) has been shown to decrease cell death due to WB (290-320 nm) exposure. A hyperthermic " 25 approach has been shown to be effective on murine bone marrow cells. Wierenga et al. report that more primitive marrow stem cells are exkemely heat resistant when compared to more differenti~tecl cells. Acute (0.5-1 hour) heat exposure (40-44~ C) can be used to elimin~te the more rapidly dividing keratinocyte populations.

CA 02230263 l998-02-24 WO 97/08295 -lO- PCTrUS96/13616 ,~ltern~tively, other ~ vho~ ent~l manipulations can also provide a selection pres~
for epidermal stem cells. Specifically, hypothermia and hypoxia, as re~i~t~nre to such changes is con~i~tent with the critical i,llpolL~lce of m~in~ g the stem cell in vivo.

5 Stem Cell Enrichment bv Sortin~ Techniques: Perhaps the most elaborate methodscullc;nlly used to isolate dirr~.. .lL populations of keratinocytes involves cell sorting techniques. Using these techniques it has been reported that keratinocytes whichexpress the highest levels of a2~ 1 integrin have the highest colony forming efficiency. Fluorescence activated cell sorting (FACS) has been used in a number of 10 different systems to isolate cell populations which express unique markers. FACS is dependent on a fluorescein-conjugated marker or antibody to label specific cellswhich are to be isolated. As mentioned previously, no specific marker has yet been identified for epidermal stem cells. A recent scientific journal article has however described a population of keratinocytes which express the cytokeratin K19. These5 cells also have some of the phenotypic characteristic ascribed to epidermal stem cells.
Antibodies to cytokeratin Kl9 are commercially available and hence may be used for FACS of this population of ker~tinocytes.
Due in part to the current lack of a m~rh~ni~m for the purification of epidermal stem cells and in part to the nature of keratinocytes to differentiate when placed into culture, ex-vivo expansion of epithelial stem has not yet been described.
This problem has however been overcome in the hematopoietic system. By investig~ting the effects which several dirrt l~llt growth factors have on the growth of hematopoietic stem cells, researchers were able to define culture conditions which these stem cells in a primitive, non-differenti~te~l state. Some of these 25 growth factors included; platelet derived growth factor (PDGF), granulocyte-macrophage colony stim~ ting factor (GM-CSF) and various interleukins.

W O 97/08295 -11- PCT~US96/13616 Isolation of epidermal stem cells followed bv expansion: Isolation of epiclPrm~
progenitor cells may be followed by limited expansion of these cells prior to application to the dermal matrix. The aim is to induce these cells to divide so as to increase the number of progenitor cells available for seeding onto the acellular dermal matrix.
Little is currently known about the definitive switch mech~ni~m which induces k~r~tinocytes to enter the cu.. iL(e(l state of ttorrnin~l differentiation. There are however some in~t~nceS in the hematopoietic system which may provide insight to this phenomenon. Specific growth factors have been found to be necessary for theo m~int-on~nce of hematopoietic stem cell culture including, PDGF, GM-CSF and various interleukins. Combinations of these factors have been used to expand hematopoietic stem cells in culture.
The epidermis has become recognized as one of the most active secretory tissues of the body. Keratinocytes have been found to secrete interleukins -1, -3, -6, -15 7, -8 and -10, colony stim~ ting factors granulocyte-colony stimlll~ting factor (G-CSF), macrophage-colony stimlll~tin~ factor (M-CSF) and GM-CSF, arachidonic acidmetabolites, metabolites of vitamin D3, p~aLhyluid hormone-related protein, collagenases, tissue inhibitor of metalloproteinases and tissue pl~cminogen activator, transforming growth factor-alpha (TGF-a), TGF-~, turnor necrosis factor- alpha 20 (I NF-a), PDGF and intracellular adhesion molecule-1 (ICAM- l). This is still only a partial list, and hip~hlight~ the complexity of growth regulation in the epicl~rmi~.
Primary keratinocytes exhibit a finite life span in vitro. Culturing conditions optimized to retain the epidermal stem cells theoretically would allow indefinite culture and expansion of these cells. In a practical sense for clinical use, it is " 25 beneficial to seed keratinocytes onto the acellular dermal matrix as quickly as possible. In order to facilitate this, a stim~ tory signal which induces the stem cell to divide once or twice will have a dramatic effect on the final expansion ratio.

W O 97/08295 -12- PCT~US96/13616 Isolated epidermal stem cells can be cultured in medium cont~ining one or more of the following growth factors which have been shown to stim~ te keratinocyte growth: platelet derived growth factor (PDGF), granulocyte-macrophage colony stim~ ting factor (GM-CSF) (both found to be nt~c~c.c~ry for hematopoietic stem cell growth), tumor necrosis factor-alpha (TNF-a), transforming growth factor-alpha (TGF-a) (both potent stimulators of keratinocyte growth) and keratinocyte growth factor (KGF). KGF, one of the more recently defined growth factors in theepicl~rmic, is a novel member of the fibroblast grovvth factor family and has been shown to have a stim--l~tory effect on keratinocyte growth. These culture conditions o may also include growth of the cells on a 3T3 fibroblast feeder layer.

PRODUCTION OF A RECONSTITUTED COMPOSITE SKIN USING
DISSOCIATED E~ERATINOCYTES IN COMBINATION WITH AN INTACT
ACELLULAR DERMAL MATRIX

In the l~cr~ d embodiment of this invention, lceratinocytes isolated from a biopsy of fresh human skin are applied directed to an intact acellular dermal matrix which is then transplanted to a skin defect.
The biopsy of fresh skin would be transported in cell culture medium cont~inin~ 10% fetal bovine serum, penicillin and streptomycin. The tissue is kept at 4 degrees cPnti~r~(le and processed within 24 hours. The tissue is h~n~1l,od with sterile instr ~m~nt~ sectecl to remove ~xL dneous fat and tissue, and cut into strips of no greater than 4 mm in width. The skin may be deepidermized with various enzymaticagents including trypsin, Dispase, Thermolysin or ethlçnP~ mint~tetraacetic acid(EDTA). The o~Li-llu,.l method involves inc~lb~tion in Dispase II (2.4 units/ml) at 37 degrees centi r~rle for 1.5 to 2 hours with periodic vortex mixinp, followed by a 30 minute incllh~tion in 0.25% trypsin plus 1 mM EDTA also at 37 degrees centigrade.
The ~. "~I~"l is then pipetted into a sep~dL~ vial, spun down to pellet cells and resuspended in growth medium. This medium is composed of a 3 :1 lllixLule of DMEM:Ham's F-12 supplementt?d with 10% fetal calfserum, 5 g/ml insulin, 0.5 g/ml hydrocortisone, 10 ng/ml epidermal growth factor, 10 ng/ml cholera toxin, and 0 15 mM Ca++- The cells would then be seeded onto the acellular dermal matrix and transplanted to the patient.
Several combinations involving the isolation of epidermal cells, in vitro culture and seeding of the acellular dermal matrix can be accomplish including:

CA 02230263 l998-02-24 The acellular dermal matrix is transplanted to the patient days prior to seedingof epidermal cells. This allows the dermal matrix time to become revasc~ ri7~d prior to the application of epidermal cells.
II The skin biopsy can be processed as described in the plerelled embodiment 5 followed by an in vitro cllltllrinp period during which the cell numbers are increased to allow seeding of a larger area of the acellular dermal matrix prior to transplantation.
III The acellular dermal matrix is transplanted to the patient days prior to seeding epidermal cells propagated as in II above.
IV The isolated epithelial cells can be directly seeded onto the acellular dermal 10 matrix followed by culturing and expansion on the acellular dermal matrix prior to transplantation. This is accomplished by l.,hyclldLillg the dermal matrix with three washes of Hank's bal~nced salt solution (HBSS), and placing the matrix in a culture flask or dish with the b~cement membrane facing up. Isolated human keratinocytesare then seeded onto the dermal matrix (at approximate S X 104 cells per cm2 of the 15 dermal matrix) and allowed to stand nn~ tllrbed in a cell culture in~ h~tQr for 24 to 48 hours before ch~n~ing the media. After this period the medium is changed withfresh medium co.,~ g 10-7 M all trans-retinoic acid. After an additional 16-24hours the medium is changed again with the further addition of 10-5 M +/-isoplolelcnol. After an additional 24 hours exposure there will be a confluent layer of 2 o keratinocytes across the dermal matrix. The composite graft can be transplanted at this point.
V The composite graft can be continued in ex vivo culture and raised to the air-liquid interface by the use of a raised culture surface (such as a metal screen) which allows medium to reach the composite only from below. This exposure of the upper2 5 surface of the graft will induce some of the keratinocytes to begin a program of dirr~ iation resulting in the formation of a stratified epiclerrni~. During the air-liquid culture period the medium can be supplemt?nte~l with other chemicals or agents which have also been shown to induce stratification in epithelial cultures. These agents in~ lc7 but are not limited to, calcium chloride and sodium butyrate. Ther~?sllltinp composite graft will now contain a fully stratified epidermis. The composite may be transplanted at this point.
VI .~lt~ tively, the epithelial cells can be cultured to produce a CEA sheet prior 5 to application to the dermal matrix. This process involves culturing of isolated keratinocytes to or exceeding confluence, at which time they will forrn an intact sheet of keratinocytes. This sheet can then be released from the culture vessel by treating with enzymes such as Dispase which disrupt the ~tt~hmPnt of cells to the substrate but do not disturb cell-cell contacts. The sheet of CEA can be transferred to the 10 acellular dermal matrix using a carrier such as Vaseline gauze followed by transplantation.
VII The acellular dermal matrix is transplanted to the patient days prior to theapplication of CEA sheets produced as in VI above.

MICROSKIN GRAFTING IN COMBINATION WITH AN ACELLULAR
DERMAL MATRIX

In the p-efell- d embodiment of this invention, a small piece of autologous, split-thickness, fresh human skin is passed through a skin mesher, fitted with acontinuous cutting blade wheel, 2 times, at a 90~ angle to each pass. Alternatively the skin can be cut into small pieces of approximately 1-1.5 mm2 using a sharp scalpel.
These microskin pieces are then spread evenly across an area of the b~ment membrane surface of the acellular dermal matrix which is approximately 10-50 times the original area of the starting piece of skin. The composite graft is then transplanted to the wound surface and covered with a sheet graft of ~;lyo~.~s~ ed, human allograft skin.
Several combinations of microskin grafting in combination with the acellular dermal matrix can be accomplished including:
5 I The microskin pieces are "dl,~rel.ed to the acellular dermal matrix which has been transplanted to the patient days previously.
II The composite of the p,efel,~d embodiment is allowed to propagate in ex vivo culture at the air-liquid interf~ e as described in EXAMPLE 1 prior to transplantation.
III The composite of the p,~er. .,~ d embodiment is composed of microskin pieces10 derived from an allogeneic skin biopsy.
IV The allogeneic microskin pieces are transferred to the acellular dermal matrix which has been transplanted to the patient days previously.
V The composite in III is allowed to propagate in ex vivo culture at the air-liquid int~rf7.ce as described in EXAMPLE 1 prior to transplantation.
VI The composite of the ~,eft;"~ d embodiment and those described in I-V are covered with a synthetic polymer membrane which is then overlaid with the opl~:served, human allograft skin at the time of transplantation.

SELECTION OF EPITHELIAL CELLS WHICH EXHIBIT CHARACTERISTICS
OF EPITHELIAL PROGENITOR OR STEM CELLS

In the pl~er~ l,.,d embodiment ofthis invention, epidermal stem cells would be isolated by using the ~rcll~ r dermal matrix as a parming substrate. This takes advantage of the previously described char~l tt?ri~tic of the putative epidermal stem cell to attach rapidly to type IV collagen. The b~ment membrane of the acellular dermal matrix is composed primarily of type IV collagen. The entire epithelial cell suspension is incubated on the derrnal matrix for 30 minlltec. The matrix is then washed with a light skeam of culture medium to wash away nn~tt~rhed cells and then transplanted onto the patient.
s There are several configurations of this grafting scenario involving stem cell isolation, propagation, see~lin~ of the acellular dermal matrix and transplantation.
These techniques may include any of the following:
Panning on the acellular dermal matrix as described in the ~ r~ d embodiment followed by release of the ~tt~rhl-A cells from the dermal matrix using o trypsin and seeAing onto acellular dermal matrix which has been transplanted onto the patient days previously.
II Isolation of the epi~lPrm~l progenitor cells by nn~rh~nicm~ which s~Jdldle due to differences in cell size, followed by seeding of the selected cells onto the acellular dermal matrix and transplantation.
III Isolation of the epiderrnal progenitor cells by mrrh~ni~m~ which separate due to selective ~tt~rhment to culture dishes coated with various dermal matrix col~lpollents (e.g. fibronectin, type I collagen, vitronectin, or various glycosaminoglycans), followed by release of the cells *om the culture dish usingtrypsin, seeding onto the dermal matrix, and transplantation.
IV Isolation of the epidermal progenitor cells by m~çh~ni~m~ which separate due to selective killing of rapidly dividing cells using antiproliferative agents, followed by seeding of the selected cells onto the acellular dermal matrix and transplantation.
V Isolation of the epidermal progenitor cells by m~rh~nicmc which separate due to selective sorting of cells t;~res~illg specific markers, followed by seeding of the acellular dermal matrix and transplantation.
VI Isolation of cells using any of the meçh~nicm~ described in II-VI followed bysee~1ing of acellular dermal matrix which has been transplanted days previously.

VII Isolation of cells using any of the mech~ni~m~ listed in II-V followed by exvivo propagation of the cells prior to seeding of the acellular dermal matrix and transplantation.
VIII Isolation and propagation of cells as described in VII followed by seeding onto acellular dermal matrix which has been transplanted days previously.
IX Use ofthe me-~h~ni~m described in VII whereby the cells are isolated, seeded onto the acellular dermal matrix and prop~g~t~rl on the dermal matrix prior to transplantation.

Claims (24)

CLAIMS:

1. A method for producing composite skin comprising a dermis inoculated directly with mammalian cells.

2. A method in claim one, whereby said dermis is acellular and intact.

3. A method in claim one, whereby cells are delivered to the dermis as a micromeshed autograft or allograft.

4. A method in claim one, whereby cells are disaggregated from epidermis and applied to the dermis and transplanted.

5. A method in claims one and three whereby the cells are delivered to the dermal matrix which has been transplanted onto the patient days previously.

6. A method in claims one and four, whereby cells are disaggregated and preselected for properties consistent with a progenitor or stem cell prior to application to the dermal matrix and then transplanted.

7. A method in claim six whereby the cells are applied to an acellular dermal matrix which has been transplanted to the patient days previously.

8. A method in claim six, whereby such properties include one or more selected from; adhesiveness of cells, cell size, label retention (long cell cycle) high clonogenic activity, expression of certain cell markers, resistance to anti-mitotic chemotherapeutic agents, and resistance to thermal variations.

9. A method in claims four to eight, whereby cells are propagated in vitro prior to application to the dermis and transplanted.

10. A method in claim nine, whereby the cells are applied to an acellular dermal matrix which has been transplanted to the patient days previously.

11. A method in claims four to eight, whereby cells are applied to the dermis and then cultured in vitro on the dermal matrix prior to transplantation.

12. A method in claims six and eight, whereby cells are propagated in vitro and then applied to the dermal matrix and cultured further prior to transplantation.

13. A method in claims nine to twelve, whereby such expansion is achieved using one or a combination of stem cell specific factors selected from a group including Granulocyte Macrophage - Colony Stimulating Factor, keratinocyte growth factors, serum factors, Transforming Growth Factor - .alpha. and .beta. and Platelet Derived Growth Factor.

14. A method in claims one to thirteen, whereby cells have been genetically modified ex vivo.

15. A method in claim fourteen, whereby such genetic modification is selected from a group consisting of one or more of the following:
- to secrete a locally acting factor;
- to secrete a systemically acting factor, engineered to overcome a specific cell defect;
- engineered to bypass a specific immune response - to exhibit transient expression - to exhibit stable expression

16. A method in claim eleven, whereby said culture is further manipulated to produce a multilayered, stratified epidermis prior to transplantation.

17. A method in claims one to sixteen, whereby the dermal matrix is of xenogeneic origin.

18. A method in claims one to sixteen, whereby the dermal matrix is of human origin.

19. A method in claims one to sixteen, whereby the cells are of xenogeneic origin.

20. A method in claims one to sixteen, whereby the cells are of human origin.

21. A method in claim twenty, whereby the cells are either autologous or allogeneic in origin or a combination of both.

22. A method in claim twenty-one, whereby said inoculated dermal matrix is applied to a person.

23. A method in claim twenty-two, whereby said application of all inoculated acellular dermis takes place without prior propagation of cells in culture.

24. A method in claim twenty-two, whereby said inoculated dermal matrix is utilized ex vivo as a laboratory assay.