Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
  • C12N5/0698—Skin equivalents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/60—Materials for use in artificial skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
  • C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1323—Adult fibroblasts

Definitions

• the present invention relates to a living chimeric skin replacement.
• This chimeric skin promotes improved wound healing.
• the compositions and o methods of this invention effect wound closure by providing a permanent replacement skin containing allogeneic epithelial cells cultured in vitro as well as freshly harvested autologous epithelial cells available for immediate implantation.
• the invention provides an immediate supply of cells for full coverage of a wound and includes the patients' own keratinocytes. 5
• the skin is comprised of both epidermis and dermis tissue.
• the epidermis contains both living and nonliving layers.
• the basal layer and the spinous layers are sometimes called the Malpighian layer. Its function is to generate the nonliving outermost barrier layer, the stratum corneum.
• the epidermis grows from its deepest basal layer or stratum basale.
• keratin-producing cells the pigment-producing cells which are derived from bone marrow and play an important role in the immune function of the skin.
• melanocytes the pigment-producing cells
• Langerhans cells which are derived from bone marrow and play an important role in the immune function of the skin.
• Cell proliferation occurs in the basal layer and in the lower levels of the stratum spinosum (or spinous layer), where cells are continually pushed toward the surface. 5 As epidermal cells move toward the surface, they become thinner, dehydrated, hardened and keratinized (a process called differentiation).
• stratum spinosum stratum spinosum
• SUBST ⁇ SHEET (RULE 26) actually consists of several layers of cells.
• Cells move from the stratum spinosum to the stratum granulosum, or the granular layer, which is generally 2-3 cells thick.
• stratum lucidum the transitional cell level
• the membrane structure is stabilized by the formation of disulfide cross- links between cysteine residues.
• Fully formed, flattened, hardened epidermal cells enter the stratum corneum.
• the dermis is primarily a supportive connective tissue comprising fibrous tissue containing blood vessels and branches of nerves.
• the dermis contains fibrob lasts, which produce connective tissue elements such as the extracellular matrix proteins, e.g., o collagens, fibronectin and elastin. These matrix proteins contribute to the strength and flexibility of the skin.
• the basement membrane serves, in part, to attach the epidermis to the dermis.
• the skin also contains accessory organs such as hair follicles and sweat glands. Depending on the severity of a wound, any or all of these elements of the skin can be damaged or lost.
• the autograft skin is usually meshed and stretched so that it can cover large wound areas.
• Such meshing and stretching results in extensive and undesirable mesh patterns of the healed skin, as well as permanent scarring.
• Reconstructive surgery may also be required over a period of many years.
• human cadaveric skin allograft when only small areas of unburned autograft skin are available 0 for wound coverage, human cadaveric skin allograft, either cryopreserved or fresh, is currently the standard biologic dressing for coverage of extensive excised burn wounds (Atnip and Burke, 1983, Curr Prob. Surg. 20:623-86; Pruitt and Levine, 1984, Arch. Surg. 119:312-22; Hansbrough, 1987, In: Boswick J, ed. The Art and Science of Burn Care. Rockville, Maryland: Aspen Publ. Inc., 57-63). 5 While fresh cadaveric skin allograft is the superior covering for excised wounds when autograft skin is not available, fresh skin is in limited supply. Another
• HLA class I analysis (Gielen et al, Dermatologica 175:166 (1987)), DNA fmge rinting (De Luca et al, Burns 15:303 (1989)), and Y-chromosome analysis (Burt et al, Br. Med. J. 298:915 (1989); Birain et al, 0 Br. Med. J. 298:917 (1989)) have evidenced ultimate replacement of the allograft by recipient keratinocytes.
• U.S. Patent No. 4,016,036 is directed to the serial culture of human epidermal cells such as keratinocytes where primary cultures of keratinocytes are initiated with a single-cell suspension derived from a full-thickness biopsy of a patient's skin, which is minced and treated with trypsin to disaggregate the cells.
• the cell suspension is plated in culture dishes containing culture medium with various growth factors.
• the cell suspension 0 overlies a layer of murine fibrob lasts that have been irradiated to prevent multiplication.
• these cultures are treated with trypsin and the disaggregated cells are passed to multiple new dishes, initiating secondary cultures. As they proliferate, the keratinocyte colonies continue to expand until adjacent colonies merge to form a confluent sheet of keratinocytes. 5
• U.S. Patent No. 4,304,866 further disclosure includes detaching the cultured autologous keratinocytes from the culture dish. To detach them from
• 4,299,819 was one of the first to describe a process for treating burn victims where epidermis is separated from the dermis, dissociating the epidermis into epidermal cells and growing the epidermal cells in the absence of dermal components in a tissue culture medium having a pH of about 5.6 to 5.9.
• the epidermal sheet is applied to an afflicted area on the burn victim.
• Gallico et al, New England Journal of Medicine 311(7):448-451 (1984) describe grafting of skin replacements using autologous epithelial sheets obtained by culturing small skin-biopsy samples.
• This procedure involves multiple operations where the burn victims first received human-cadaver skin allografts or collagen- glycosaminoglycan-silatic sheets while the autologous cells grow in vitro.
• the autologous skin required several weeks of growth, the sheets of autologous cells further required release of the cultured sheets from culture flasks with Dispase prior to implantation. Further, the allografts needed to be removed prior to implantation of the autologous cultures. Problems associated with this technique include decrease in body temperature and blood loss from allograft removal.
• Bell, U.S. Patent No. 4,485,096 describes skin grafts in the rat model containing fibroblasts in collagen lattices and seeded with epidermal cells previously
• SUBST ⁇ UTE SHEET (RULE 26) biopsied from the host recipient.
• the grafts are cultured in vitro and implanted at the wound site.
• the enzymatic treatment used to remove a newly formed epithelial sheet from a tissue culture plate can alter or destroy cell surface molecules, which, in turn, can alter the ability of the cultured cells to adhere to each other or to the wound.
• the multilayered cultured epidermal cells can change from a proliferating state into a differentiated state.
• associated with differentiation of the cells is a loss of expression of certain cell adhesion molecules, which, as indicated above, can alter the binding of the cells to each other and to the wound and, therefore, result in a failure to effect wound closure.
• 5,610,007 disclose generating a chimeric epithelium in vitro where 50:50 and 25:75 mixtures of Balb/c and C3H/HeN mouse keratinocytes were co-cultured to confluence and then enzymatically removed with Dispase (Sigma) to release the cells from the flask's surface.
• Dispase Sigma
• the present invention relates to a living chimeric skin replacement, methods for preparing it, and methods of using it, in the treatment of patients with skin wounds caused by a variety of conditions, including for example, lacerations, ulcers and burns.
• the invention also includes a novel composite skin replacement for use in the treatment of wounds.
• the invention is based, in part, on Applicants' discovery that an improved wound covering can be made by grafting allogeneic epithelial cells, such as keratinocytes, which have been cultured on a membrane or other biocompatible substrate in vitro and, at or about the time of grafting, adding a relatively small amount of autologous epithelial cells to the allogeneic cell-substrate construct so that the autologous epithelial cells are implanted simultaneously with the allogeneic cells.
• the autologous cells are preferably keratinocytes which are seeded onto the cultured allogeneic cells just prior to the grafting.
• the autologous cells can be seeded directly into the wound site, preferably on top of a dermal replacement construct, and the allogeneic cell-substrate construct placed, allogeneic cells facing into the wound, on top of the autologous cells.
• the allogeneic cells are cultured on a membrane or other biocompatible substrate in vitro and cryopreserved so that a fresh supply of the graft is readily available to the surgeon.
• the allogeneic cells are cultured on a biocompatible, biodegradable membrane which is reversibly hydratable.
• the autologous cells are added to the allogeneic graft at a density of about 1 x 10 4 cells/cm 2 of graft material.
• DERMAGRAFT Advanced Tissue Sciences, Inc., La Jolla, CA
• DERMAGRAFT Advanced Tissue Sciences, Inc., La Jolla, CA
• DERMAGRAFT is a living dermal replacement comprised of a three-dimensional living stromal tissue comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to, and substantially enveloping a biocompatible framework or scaffolding.
• DERMAGRAFT is described in detail in United States Patent No. 4,963,489, the disclosure of which is incorporated by reference herein in its entirety.
• a composite skin replacement is utilized o according to the methods of this invention, wherein a biocompatible dermal construct with a biodegradable or removable scaffolding as a base, such as DERMAGRAFT, is placed into a wound bed, autologous cells from the patient are added on top of the dermal construct, e.g., immediately or after the dermal construct has had time to vascularize, and then an outer component comprising allogeneic epithelial cells cultured on a dermal component 5 comprising a biocompatible dermal construct with a biodegradable or removable scaffolding as a base, combined with a transitional covering such as a BIOBRANE membrane, is placed on top of the autologous cells.
• a biocompatible dermal construct with a biodegradable or removable scaffolding as a base such as DERMAGRAFT
• this composite skin replacement include the use of allogeneic cells or autologous cells, or combinations of both, in the middle or outer components of the composite.
• allogeneic and autologous epithelial cells can be simultaneously inoculated onto a substrate, cultured for a sufficient time to allow for attachment of the cells to the substrate, and then implanted into a wound site, the cells facing into the wound.
• the present invention relates to a living chimeric skin replacement.
• the skin replacement of the invention is a permanent replacement comprising a chimeric construct containing allogeneic and autologous epithelial cells.
• the allogeneic epithelial cells are cultured on a substrate such as, for example, a biocompatible membrane, dermal construct, 0 glue or gel in vitro and, in most cases, cryopreserved.
• the allogeneic cell-substrate construct is then thawed (if cryopreserved) and placed at the wound site, together with autologous epithelial cells such as keratinocytes, mechanically disrupted skin or microskin bits, the autologous cells having been biopsied, preferably just prior to grafting.
• the epithelial replacement may be grafted with a permanent dermal replacement, if the 5 wound is deep.
• the present invention provides for an improved skin replacement in that an immediate skin covering of allogeneic cells cultured in vitro on a substrate such as a membrane or a dermal construct, is provided to the wound site together with healthy autologous epithelial cells, thereby expediting healing.
• the ratio of autologous cells to allogeneic cells can be in the range of 1:5 to 1 :50.
• the allogeneic cells provide sufficient coverage before an immunologic response can be raised at the wound site.
• the autologous epithelial cells have a physiological and immunological selective advantage at the wound and eventually replace the allogeneic cells.
• the amount of autologous cells provided is such that the wound heals with a sufficient coverage of autologous cells before a significant immunologic response occurs.
• the present invention has the advantage of being an immediate and available skin graft which, because of the added autologous cells, is a permanent graft without significant immunological rejection.
• allograft cells from cadaveric skin are inoculated with host autologous cells, and implanted at the wound site.
• the autologous cells then proliferate and grow progressively replacing the allograft tissue as the host's immunological response is raised against the cadaveric tissue.
• the allogeneic epithelial cells are cultured in vitro so that they are available for immediate grafting when necessary.
• the allogeneic epithelial cells are grown on a biocompatible, preferably biodegradable substrate.
• a living chimeric skin replacement may be produced as follows:
• allogeneic epithelial cells are plated onto a biodegradable, biocompatible substrate and are allowed to grow to about 25-90% confluence;
• step (d) autologous epithelial cells are harvested and inoculated onto the allogeneic cells at a density of about 1 x 10 4 /cm and then implanted at the wound site; and (e) the chimeric skin graft is covered with a dressing sufficient to adequately immobilize the graft, and protect it from mechanical and infectious forces. It should be noted that steps (b) and (c) of this method of the invention are optional.
• a preferred embodiment of the invention additionally provides that a dermal replacement be placed into the wound site with the subsequent application of the chimeric skin graft.
• a dermal replacement be placed into the wound site with the subsequent application of the chimeric skin graft.
• the use of a dermal replacement in deep or full thickness wounds helps to create an optional environment for the keratinocytes to attach and proliferate.
• a major problem for epithelial replacement is the availability of an adequate dermal foundation for grafting at the site of extensive wounds. It is optimal if dermal matrices are readily available.
• deep or full thickness skin wounds use a cultured dermal replacement such as DERMAGRAFT (Advanced Tissue Sciences, Inc., La Jolla, CA) to support keratinocyte attachment and proliferation.
• DERMAGRAFT is composed of allogeneic neonatal fibroblasts cultured on biodegradable polyglactin and is a permanent dermal replacement (since the polyglactin is biodegradable). No significant immune response is raised to this graft since there is little immunogenicity associated with these fibroblasts.
• a three-dimensional chimeric skin graft may be produced in deep or full thickness wounds as follows:
• allogeneic epithelial cells are plated onto a biodegradable, biocompatible substrate and are allowed to grow to about 25-90% confluence;
• a chimeric skin replacement comprising three components: (1) an inner component comprised of a biocompatible dermal construct with a biodegradable or removable scaffolding as a base; (2) a middle component comprised of autologous epithelial cells, such as keratinocytes or mechanically disrupted skin, derived from the patient or an autologous donor, and (3) an outer component comprising allogeneic epithelial cells, such as keratinocytes, cultured on a dermal construct comprising a biocompatible dermal portion with a biodegradable or removable scaffolding as a base combined with a transitional covering such as a BIOBRANE membrane.
• the allogeneic cells are grown on and attach to the dermal portion of the outer component (as opposed to the membrane covering) and the dermal side of the outer component containing the cells is facing inward toward the autologous cells of the middle component.
• This composite skin replacement can be formed in vitro for use subsequently for treating wounds, such as burns.
• this composite can be formed at the time of implantation, i.e., the inner dermal construct component can be placed into the wound site, the autologous cells added on top of the dermal component, either immediately or after the dermal component has had time to vascularize, and then the outer component with the dermal side facing inward and containing allogeneic cells is placed on top of the autologous cells.
• the outer most layer will be the membrane of the outer component, followed by the dermal portion of that component containing the allogeneic cells, followed by the autologous middle layer, followed by the inner most dermal replacement layer.
• the middle component can consist of cells from allogeneic sources or a combination of autologous and allogeneic sources.
• the cells of this middle component can be in the form of single cell suspensions, microskin bits, disrupted or dispersed skin or in sheet form.
• the cells cultured on the outer component may be autologous or a combination of autologous and allogeneic cells and they may be cultured with or without autologous or allogeneic proteins or combinations thereof.
• the outer component is a modified version of DERMAGRAFT-TC; also known as TRANSCYTE, DERMAGRAFT-TC is a dermal replacement construct comprising a non- viable dermal component attached to a sialastic membrane such as BIOBRANE (as disclosed in United States Patent No. 5,460,939, the disclosure of which is incorporated herein by reference).
• a modified version of TRANSCYTE comprises this construct with cellular or protein additions. More particularly, a preferred embodiment is this dermal replacement construct with allogeneic epithelial cells growing on it, with or without added protein factors, e.g., to aid in growth and proliferation.
• This outer component serves several purposes. It provides a good barrier to control moisture loss and prevent infection. It also holds all of the other components in place, allowing them the best opportunity to adhere to the wound site in an environment that optimizes their growth. Furthermore, it is translucent and thus allows for visual observation during the healing process.
• Both the inner component and the outer component can be grown or formed in vitro and cryopreserved and stored for subsequent application in vivo.
• a non- viable DERMAGRAFT is placed into a wound bed with mechanically dispersed or disrupted autologous skin from the patient placed on top and covered with a piece of TRANSCYTE that has been modified by culturing allogeneic keratinocytes on one aspect, preferably on the side facing into the wound, and/or coating with proteinaceous solutions.
• the patient's skin cells grow to confluence atop the inner DERMAGRAFT and then the outer component is eventually removed or may slough off.
• the substrate should be biocompatible and preferably biodegradable so that the substrate degrades over time and does not need to be removed at a later date.
• the substrate such as a membrane substrate for example, should be one which is reversibly hydratable, so that it allows for absorption and/or release of exudate in order to prevent the accumulation of exudate at the wound site, and prevents desiccation of the wound.
• the membrane preferably has a small pore size so that it reduces or inhibits the ability of bacteria to infect the wound.
• the substrate should readily adhere to the wound or be capable of being treated with a bioadhesive so that adherence to the wound occurs without harming the epithelial cells or significantly changing the moisture vapor transmission (MVTR) of the membrane.
• the substrate should also be adequately malleable so that it conforms to variable wound surfaces.
• the allogeneic epithelial cells are grown on a synthetic hydrophilic polyurethane membrane dressing, such as, for example, HYDRODERM (Wilshire Medical, Inc., Dallas, TX) or SPYROFILM (Polymedica Inc., Denver, CO).
• HYDRODERM Wang Medical, Inc., Dallas, TX
• SPYROFILM Polymedica Inc., Denver, CO
• Both films have high but variable water vapor permeability and a biocompatible adhesive coating which covers a portion of the membrane surface. When they become wet, these polyurethane membranes significantly increase their water-vapor permeability.
• the MVTR of HYDRODERM is a function of the degree of wetness of the membrane. Normal skin has a range of MVTR of 200-2000 g-m "2 -d " '. Damaged or burned skin may have moisture transmission as high as 3000-5000 g-m 2 -d " '.
• SUBST ⁇ SHEET 1500-2000 g-m ⁇ -d "1 .
• the MVTR falls to 500-1000 g-m "2 -d " ' because the adhesive blocks water vapor transmission.
• the MVTR should not be one that allows for maceration of tissues, channeling, and leakage and potentiates infections.
• MITRAFLEX PolyMedica Industries, Inc.; Golden CO which is a flexible, transparent polyurethane membrane that allows moisture vapor to be transmitted but is impermeable to other liquids and to microorganisms (see Reed, J. Biomat. Appl. 6:26-31 (1990)).
• the cultured allogeneic epithelial cells may be inversely applied to the
• Keratinocytes cultured on these membranes have been shown to highly express integrins involved in the wound healing processes.
• Hyaluronic acid membranes may also be used for culturing the keratinocytes.
• Hyaluronic acid has been shown to have the properties of biocompatibility and low
• Another embodiment of the invention is the use of fibronectin mats.
• Ejim et al. reported the use of fibronectin mats as an in vitro support for keratinocytes (Ejim et al, Biomaterials 14:742 (1993)). Furthermore, it was shown that fibronectin inhibits differentiation of cultured keratinocytes. Adams et al, Cell 63:425 (1990). Keratinocytes
• Another substrate for culturing the allogeneic cells is a dermal construct
• the allogeneic cells can be applied to either aspect or side of the dermal construct, but preferably are applied to the dermal aspect.
• the allogeneic cells can be cultured with or without additional protein factors applied to the dermal construct.
• Another substrate for culturing the allogenic epithelial cells of the invention is fibrin glue or a collagen gel.
• Any membrane or substrate capable of supporting the growth of cultured epithelial cells may be used in the invention.
• the cells, once cultured on the membrane or substrate, do not have to be enzymatically removed.
• hydrogel which is a polymer composition, preferably prepared from hydrophihc polymers, wherein the polymer, natural or synthetic, is cross-linked, e.g., via photopolymerization, to create a three-dimensional lattice structure which traps water molecules to form a gel.
• Polymers useful for forming hydrogels include polysaccharides or carbohydrates, such as alginates, polyphosphazines, polyethylene glycol or polyethylene oxide.
• the hydrogels used herein are polymerized prior to the addition of the allogeneic cells. Details for preparing hydrogels for use in this invention are set forth in the United States application Serial No. 08/862,740, filed May 23, 1997, the disclosure of which is incorporated herein by reference in its entirety.
• Allogeneic epithelial cells may be cultured on the membranes and substrates of the invention by any appropriate means known in the art, such as, for example, the methods contained in Doyle, A., Griffiths, J.B., and Newell, D.G., "Cell & Tissue Culture: Laboratory Procedures," Wiley Publishers (1995).
• keratinocytes and melanocytes may be isolated as follows.
• a tissue sample, ej*. foreskin, may be trimmed so that the entire surface may be easily exposed to antibiotics.
• subcutaneous tissue is removed with sterile scissors and approximately 10 ml PBS with Ca++, Mg++ is added to the dish to rinse off cellular debris.
• the tissue is then washed in a concentrated antibiotic solution and let sit for 5 minutes.
• the tissue is then transferred to 0.25% trypsin/EDTA, cut lengthwise into strips or squares approximately 3 mm wide with sterile scalpel or scissors.
• the cut tissue in trypsin is placed in a CO2 incubator at 37 °C, 5% C02, 90% humidity for 2.5-3 hrs.
• the tissue pieces may be removed from the trypsin solution, and the epidermis separated from the dermis using curved forceps.
• the epidermis may be placed in a conical tube, and about 0.15% trypsin in calcium-magnesium free PBS, may be used to digest the tissue into a single cell suspension; to facilitate this process, the sample may be repeatedly
• the flasks are placed in a CO2 incubator at 37 °C, 5% C02, 90% humidity and the cells are mitotically expanded.
• These allogeneic epithelial cells are then applied to a substrate, e.g., a membrane in a petri dish, and allowed to grow, e.g., at 37 °C, 5% C02, 90% humidity.
• a substrate e.g., a membrane in a petri dish
• the cells are either cryopreserved or used for implantation.
• the cultured epithelial sheets may be cryopreserved by methods known in the art. See, for example, the methods described in Tubo et al, U.S. Patent No. 5,145,770 which is incorporated by reference.
• the allogeneic cells on the substrate are then thawed at the appropriate time, generally just prior to grafting.
• the allogeneic cells can be genetically engineered prior to being cultured on the substrate to "knock out” expression of factors that promote patient immunological responses against the implanted allogeneic cells. Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels are discussed below. "Negative modulation”, as used herein, refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene product in the absence of the modulatory treatment.
• a gene native to the allogeneic cell can be reduced or knocked out using a number of techniques, for example, expression may be inhibited by inactivating the gene completely (commonly termed "knockout") using standard homologous recombination techniques.
• a positive selectable marker for example ne ⁇
• a gene may also be inactivated by creating a deletion in part of a gene, or by deleting the entire gene.
• SUBST ⁇ UTE SHEET (RULE 26) activity For example, antisense RNA molecules which inhibit the expression of major histocompatibility gene complexes (HLA) have been shown to be most versatile with respect to immune responses.
• appropriate ribozyme molecules can be designed as described, e.g., by Haseloff et ah, 1988, Nature 334:585-591; Zaug et al.. 1984, Science 224:574-578; and Zaug and Cech, 1986, Science 231 :470-475.
• triple helix molecules can be utilized in reducing the level of target gene activity.
• the expression of MHC class II molecules can be modified in the allogeneic cells of this invention in order to reduce the risk of rejection of the chimeric implant.
• the histocompatibility of the implanted allogeneic cells can be enhanced or improved if, after undesirable MHC antigens have been knocked out, one or more genes encoding one or more recipient-compatible HLA antigens are transfected into the allogeneic cells prior to implantation.
• the antigenicity of the allogeneic cells can be modified to improve histocompatibility of the cells with the recipient patient.
• Autologous epithelial cells are isolated, preferably immediately prior to transplantation by the following method. Healthy tissue from the autologous donor is washed with 70% Ethanol, cold proviodine, and then 70% Ethanol. The washed tissue is dissected to remove the epidermal layer from the dermal layer. The epidermal layer is then washed in cold Phosphate Buffered Saline and suspended in a 0.25% trypsin-EDTA solution with 2.5 mg/ml dispase and mechanically disrupted for 10 minutes at room temperature. The epithelium pellet is resuspended in PBS at an appropriate concentration.
• the autologous cells are then added to the cultured allogeneic cells on the surface of the substrate described supra or alternatively, implanted directly into the wound site and then covered with the allogeneic cell-substrate construct which is inverted into the wound, cells facing into the wound as described herein.
• a cultured dermal skin replacement such as DERMAGRAFT is placed into the wound bed.
• the DERMAGRAFT may be cryopreserved and thawed prior to use, in which case, it is rinsed in NaCl solution (e.g., 0.9% normal saline).
• NaCl solution e.g. 0.9% normal saline.
• the wound site is preferably cleaned/sterilized prior to
• SUBST ⁇ UTE SHEET (RULE 26) implantation by scrubbing or rinsing with a sterilizing solution, e.g., iodine surgical scrub (e.g., 0.75% titratable iodine) or 0.9% sodium chloride solution.
• a sterilizing solution e.g., iodine surgical scrub (e.g., 0.75% titratable iodine) or 0.9% sodium chloride solution.
• the autologous cells adhere to the surface of the allogeneic cell-substrate construct (i.e., the cells adhere to the surface of the construct containing the allogeneic cells) and the construct is inverted and placed into the wound site, i.e., with the cells facing into the wound and toward the DERMAGRAFT.
• the wound site is then covered with a protective dressing or may be closed and sealed with sutures.

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• 1999
  • 1999-02-23 KR KR1020007009299A patent/KR20010072553A/ko not_active Application Discontinuation
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  • 1999-02-23 EP EP99936092A patent/EP1062322A2/de not_active Withdrawn
  • 1999-02-23 JP JP2000533527A patent/JP2002504412A/ja active Pending
  • 1999-02-23 WO PCT/US1999/003859 patent/WO1999043787A2/en not_active Application Discontinuation
  • 1999-02-23 CA CA002324452A patent/CA2324452A1/en not_active Abandoned
  • 1999-02-23 AU AU33077/99A patent/AU760470B2/en not_active Ceased

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