Patent 6,943,024

The present invention provides methods and compositions for providing graft recipients with epidermal melanocytes. Specifically, the methods and compositions of the invention provide for populations of epidermal melanocytes that may be isolated from a patient and cultured in vitro to generate a proliferating population of epidermal melanocytes that exhibit growth, migration and melanin production. The invention is based on the discovery of a culture medium composed of natural components, for culturing epidermal melanocytes that exhibit increased proliferative capacity, migratory behaviors and melanin production. The invention provides novel in vitro methods for culturing epidermal melanocytes, including those isolated from the skin of a healthy subject, to generate a proliferating population of epidermal melanocytes. The methods and compositions of the invention may be used for transplantation to treat patients having hypopigmentation or depigmentation skin disorders.

SUMMARY OF THE INVENTION

The present invention provides novel compositions and methods for culturing epidermal melanocytes. The methods and compositions of the present invention may be used to provide skin pigmentation to patients having skin disorders characterized by a lack of skin pigmentation. Such disorders include but are not limited to vitiligo or loss of skin pigmentation due to skin diseases such as inflammation, chemical or physical damage. In addition, the methods of the invention may be used in combination with dermabrasion techniques for cosmetic purposes.

The compositions of the invention relate to a culture medium comprising a basal medium, supplemented with serum, growth factors, and agents designed to elevate cAMP levels. In addition, the present invention relates to compositions comprising epidermal melanocytes cultured in a medium comprising a basal medium, supplemented with serum, growth factors and agents designed to elevate cAMP levels.

The present invention further relates to methods of culturing epidermal melanocytes for transplantation using the medium of the present invention to obtain a proliferating population of epidermal melanocytes that is capable of migratory behavior and melanogenesis. The invention is based on the observation that epidermal melanocytes cultured in the medium of the present invention possess quantitatively enhanced proliferation, migration and melanogenesis capabilities.

The present invention provides grafting methods of treating skin pigmentation disorders utilizing cultured epidermal melanocytes, comprising (1) isolating epidermal melanocytes from an individual, (2) culturing the epidermal melanocytes in the medium of the invention to obtain an expanded population of epidermal melanocytes with enhanced proliferative, migratory and melanogenesis capabilities, and (3) transplanting the expanded culture of epidermal melanocytes onto the individual's skin.

In a specific embodiment of the invention, cultured epidermal melanocytes may be genetically engineered, prior to transplantation, to enable them to produce a wide range of proteins, including but not limited to, growth factors, cytokines, extracellular matrix proteins, or other biologically active molecules. In this way, any new tissue derived from the transplanted epidermal melanocytes will produce the desired biologically active protein.

The present invention, which relates to a culture medium system with natural and physiological components, provides a more physiological micro-environment for incubation of epidermal melanocytes. In addition to serving as a source of cells for transplantation, the culture medium system of the invention provides an in vitro model system for testing the effects of various biological substances (e.g., drugs, herbs and cosmetics, etc.) on the growth, melanogenesis, migration ability and other functions of epidermal melanocytes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compositions and methods for culturing epidermal melanocytes and the use of such melanocytes in skin grafts. Specifically, the methods and compositions of the invention may be used to treat skin pigmentation disorders resulting from destruction of epidermal melanocytes or loss of epidermal melanocyte functions. The compositions of the invention relate to a culture medium comprising basal medium, supplemented with serum, growth factors, and cAMP-elevating agents. The medium of the invention differs from previously described medium in that it contains only natural, physiological agents. The present invention is based on the observation that epidermal melanocytes isolated and cultured in the medium of the present invention have enhanced proliferation, migration and melanogenesis.

5.1 Isolation and Culturing of Epidermal Melanocytes

Epidermal melanocytes may be obtained from a variety of different donor sources. In a preferred embodiment, autologous epidermal melanocytes are obtained from the subject who is to receive the melanocytes. This approach is especially advantageous since the immunological rejection of foreign tissue and/or a graft versus host response is avoided. In yet another embodiment of the invention, allogenic epidermal melanocytes may be obtained from donors who are genetically related to the recipient and share the same transplantation antigens on the surface of their melanocytes. Alternatively, if a related donor is unavailable, melanocytes from antigenetically matched donors may be used. Furthermore, because melanocytes do not express HLA antigen even allogenic melanocyte transplantation is possible.

Epidermal melanocytes may be obtained from the skin using a variety of different methods. The cells are derived from pigmented areas of the body and preferably from the subject in need of the skin graft. Epidermal melanocytes may be obtained from a superficial skin biopsy. One or more shave biopsies of the skin are taken from normal skin area after local anesthesia with 0.5%-1% lidocaine. Various skin graft instruments (e.g., a skin graft knife with a razor blade) can be used to take the biopsy (Loentz et al., 1994, J Am Acad Dermatol 30:591).

Epidermal melanocytes may also be obtained from a suction blister. Blisters may be produced by applying a vacuum of 300-400 mmHg to the surface of the skin for 30-90 minutes. The blisters may be 100-250 mm². The tops of the blisters are then excised and processed for culturing (Chen et al., 2000, J Dermatol 27:434). Alternatively, blisters may be produced by freezing with liquid nitrogen (Suvanprakon et al., 1985, J Am Acad Dermatol 13:968).

Epidermal sheets may be dissociated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells, making it possible to disperse the individual cells (Chen et al., 2000, J Dermatol 27:434). Enzymatic dissociation can be carried out by treating the skin tissue with any of a number of digestive enzymes. Such enzymes include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase and/or hyaluronidase. Chelating agents include, but are not limited to, EDTA. The basal layer may be teased away gently with forceps to dissociate cells from the tissue.

The cell suspension may be rinsed in medium or buffer and centrifuged one or more times to remove dissociated cells from tissue. Following preparation of a single cell suspension, the cells are cultured in the medium of the invention. In a preferred embodiment of the invention, the cell suspension may be grown in culture to inhibit the growth of keratinocytes and fibroblasts by the addition of geneticin (Halaban et al., 1984, In Vitro 20:447).

The present invention relates to a novel culture medium, referred to herein as Hu74 Medium, for use in culturing epidermal melanocytes. The medium of the invention differs from previously described medium in that it contains only natural, physiological agents.

The media of the invention comprises basal medium supplemented with serum, growth factors, and cAMP elevating agents. The basal medium may be any of the standard culture medium that provides the minimal requirements to sustain the growth of cells in culture. Such basal media, include but are not limited to basal amino acid/salt mixtures such as Ham's F12, RPMI, or DMEM. Serum is added to the media in concentrations of approximately 5-30%. Any type of animal serum may be used, including but not limited to, fetal calf, calf or human serum. Additional additives to the medium may include, for example, glucose, glutamine, vitamins and any additional additives known to those of skill in the art.

Growth factors and cytokines to be added to the basal medium include hepatocyte growth factor (HGF) and fibroblast growth factors (FGFs) such as bFGF. The present invention is based on the observation that HGF is a potent stimulator of epidermal melanocyte migration. This effect can induce transplanted epidermal melanocytes to migrate to the margin of transplanted area, which is important for decreasing the hypopigmentation at the margin of the transplanted area in patients.

Furthermore, the addition of both HGF and bFGF to the medium was found to enhance the proliferation of epidermal melanocytes indicating that HGF has an additive growth stimulating effect in conjunction with the bFGF. Thus, addition of HGF to the medium of the invention can further stimulate the growth of epidermal melanocytes in vitro, thereby shortening the patient's waiting time period for the transplantation. Further, in patients wherein epidermal melanocytes do not grow well and where the in vitro expansion of cell number required for transplantation cannot be met, HGF can be added to improve the growth of cells thereby facilitating transplantation.

In an embodiment of the invention, the culture medium of the invention contains bFGF. In a specific embodiment of the invention, the concentration of bFGF to be added to the media is between 1 and 1000 ng/ml. In a preferred embodiment of the invention, the concentration of bFGF in the media is between 10 and 100 ng/ml. In a more preferred embodiment of the invention, the concentration of bFGF in the media is between 20 and 50 ng/ml.

In an embodiment of the invention, the culture medium of the invention contains HGF. In a specific embodiment of the invention, the concentration of HGF to be added to the media is 10 to 1000 ng/ml. In a preferred embodiment of the invention, the concentration of HGF in the media is 50 to 750 ng/ml. In a more preferred embodiment of the invention, the concentration of HGF in the media is 100-500 ng/ml. In a specific embodiment of the invention, the Hu74 Medium of the invention contains HGF at a concentration of 100 ng/ml.

In addition, the present invention relates to Hu74 media comprising both HGF and bFGF. Addition of HGF to culture medium containing bFGF has been shown to have an additive stimulating effect on epidermal melanocytes in vitro.

The medium of the invention further comprises, one or more natural, non-toxic, physiological agents capable of promoting epidermal melanocytes proliferation in culture through cellular elevation of cAMP. As described herein, the use of epinephrine and α-MSH in cAMP deleted medium stimulated the growth, melanogenesis and migration of epidermal melanocytes.

In an embodiment of the invention, the concentration of αMSH in the media is between 1 and 1000 ng/ml. In a preferred embodiment of the invention, the concentration of αMSH is between 50 and 750 ng/ml. In a more preferred embodiment of the invention, the concentration of αMSH in the media is between 75 and 500 ng/ml. In a specific embodiment of the invention, Hu74 Medium contains αMSH at a concentration of 100 ng/ml.

In an embodiment of the invention, the concentration of epinephrine to be added to the media is between 1 and 3000 ng/ml. In a preferred embodiment of the invention, the concentration of epinephrine in the media is between 500 and 2000 ng/ml. In a more preferred embodiment of the invention, the concentration of epinephrine in the media is between 750 and 1750 ng/ml. In a specific embodiment of the invention, the concentration of epinephrine is 1.5 μg/ml. Because epinephrine is not very stable and is easily oxidized, antioxidants such as ascorbic acid (5 μg/ml) may be added to the Hu74 medium to stabilize the epinephrine.

In a specific non-limiting embodiment of the invention Hu74 Medium is prepared as follows. Glutamine is added to Ham's F12 (GIBCO, Carlsbad, Calif.) at a concentration of 2 mM. Gentamicin is added to the culture medium to obtain a final concentration of 50 μg/ml. Fetal bovine serum (FBS, GIBCO, Carlsbad, Calif.) is added to the medium to obtain a 10% concentration (volume/volume). The medium containing FBS and gentamicin is stored at 4° C. and prepared fresh every two weeks. bFGF (PeproTech, Rocky Hill, N.J.) is dissolved in F12 medium to 2,500 ng/ml and stored in small vials at -70° C. The stored solution is added to the culture medium to obtain a final concentration of 25 ng/ml once a week. HGF (PeproTech, Rocky Hill, N.J.) is dissolved in F12 medium to 10,000 ng/ml and stored in small vials at -70° C. The stored solution is added to the culture medium to obtain a final concentration of 100 ng/ml once a week. An α-MSH solution is obtained by dissolving the α-MSH powder (Sigma, St. Louis, Mo.) to 100 μg/ml and is stored at -70° C. The stored solution of α-MSH is added to the culture medium to obtain a final concentration of 100 ng/ml once a week. (L)-Epinephrine bitartrate (Sigma, St. Louis, Mo.) is dissolved in PBS at 0.15 mg/ml with sodium ascorbate (Sigma, St. Louis, Mo.) (0.5 mg/ml) and stored at -70° C. Ephinedrine and ascorbic acid are added to the culture medium to obtain a final concentration of 1.5 μg/ml of epinephrine and 5 μg/ml sodium ascorbate once a week.

Those of skill in the art will also recognize that one or more commercially available substances may be used as additives or substitutions to the medium to support the growth of epidermal melanocytes. Such growth may be monitored using a number of different methods. For example, proliferation of cells can be monitored by cell counts using a hemocytometer or flow cytometer; cell migration can be measured in chemotaxis assays using, for example, a Boyden chamber; and production of melanin can be measured using spectrophotometry.

5.2 Administration of Epidermal Melanocytes

In a specific embodiment of the invention, the cultured epidermal melanocytes of the invention are administered to a subject in need of skin pigmentation. Cultured epidermal melanocytes may be grafted directly onto the recipient where the cells will proliferate and migrate to form new skin tissue.

Prior to transplantation of cultured epidermal melanocytes, the epidermis of the depigmented or hypopigmented area of skin is removed down to the dermal-epidermal junction by superficial abrasion, which can be obtained by (i) mechanical methods, e.g., abraded at 25,000 revolutions per minute with a dermabrader fitted with diamond fraizes (Loentz et al., 1994, J Am Acad Dermatol 30:591-7), (ii) removal of the epidermis with a skin graft knife or laser, e.g., using a Silktouch Flashscanner attached to a Sharplan 1030 CO₂ laser at the setting of 4.5 to 7 watts with a 0.2 second pulse duration (Chen et al., 2000, J. Dermatol 27:434), (iii) application of YAG laser (Pai et al., 2002, J Eur Acad Dermatol Venereol 16:604), or (iv) by liquid nitrogen (Gauthier et al., 1992, J Am Acad Dermatol. 26:191).

The methods of the present invention may further be used in combination with human skin resurfacing techniques, including chemical peeling, dermabrasion, laser surgery, and various other methods for cosmetic purposes or scar removal. The methods utilize chemical, mechanical and laser techniques for removal of the upper dermal layers of the skin that can cause scarring and persistent redness. In an embodiment of the invention, epidermal melanocytes are applied to the skin after a skin resurfacing treatment.

The present methods and compositions may additionally employ cultured epidermal melanocytes genetically engineered to enable them to produce a wide range of functionally active biologically active proteins including, but not limited to, growth factors, cytokines, hormones, inhibitors of cytokines, peptide growth and differentiation factors, and extracellular matrix proteins. Methods which are well known to those skilled in the art can be used to construct expression vectors containing a nucleic acid encoding the protein of interest linked to appropriate transcriptional/translational control signals. See, for example, the techniques described in Sambrook, et al., 1992, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., and Ausebel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, N.Y.

In addition, cultured epidermal melanocytes may be attached in vitro to a natural or synthetic matrix that provides support for the transplanted epidermal melanocytes prior to, during, and/or post-transplantation. The matrix will have all the features commonly associated with being biocompatible, in that it is in a form that does not produce an adverse, or allergic reaction when administered to the recipient host. Growth factors capable of stimulating the growth and regeneration of, for example, skin tissue may also be incorporated into matrices. Such matrices may be formed from both natural or synthetic materials and may be designed to allow for sustained release of growth factors over prolonged periods of time. Thus, appropriate matrices will both provide growth factors and also act as an in situ scaffolding in which the epidermal melanocytes may proliferate and migrate. In preferred embodiments, it is contemplated that a biodegradable matrix that is capable of being reabsorbed into the body will likely be most useful.

To improve epidermal melanocytes adhesion to the matrix, survival, function and/or migration of the epidermal melanocytes, the matrix may optionally be coated on its external surface with factors known in the art to promote cell adhesion, growth, survival or migration. Such factors may include cell adhesion molecules, extracellular matrix molecules or growth factors.

Epidermal melanocytes can be administered to the recipient in an effective amount to achieve its intended purpose. More specifically, an effective amount means an amount sufficient to lead to the restoration of skin pigmentation or skin tissue function, thereby alleviating the symptoms associated with disorders resulting from genetic defects or tissue damage. The progress of the transplant recipient can be determined using visual inspection of the transplant area to assess cell coverage and skin pigmentation. The number of cells needed to achieve the purposes of the present invention will vary depending on the degree of tissue damage and the size of the affected skin area. Determination of effective amounts is well within the capability of those skilled in the art.

Cultured melanocytes can be detached from the culture flask by various enzymes, such as for example, by trypsin (0.05%,-EDTA (0.02%), at 37° C. for 3-10 minutes. The cell suspension is then centrifuged and the cells are resuspended with serum free Hu74 medium and placed in contact with the denuded area of the skin surface (Chen et al., 2000, J. Dermatol 27:434-439; Olsson et al., 1992, Lancet 340:981; Olsson et al, 1995, Br J Dermatol 132:587).

Alternatively, epidermal melanocytes can be administered to the recipient in one or more physiologically acceptable carriers. Carriers for these cells may include, but are not limited to, solutions of phosphate buffered saline (PBS), lactated Ringer's solution containing a mixture of salts in physiologic concentrations or culture medium with or without serum. In addition, the cells may be associated with a biocompatible matrix prior to administration into the recipient host.

The methods of the present invention encompass grafting of epidermal melanocytes onto the skin. The grafting of the epidermal melanocytes onto the desired region on the skin is accomplished by directly applying the cell layer to the area of the skin. In addition, growth factors or hormones may be administered to the recipient prior to and following transplantation for the purpose of priming the recipients tissue to accept the transplanted cells and/or to generate an environment supportive of cell proliferation and/or differentiation and/or migration and/or melanogenesis.

The site may be covered with a protective covering such as silicone (Chen et al., 2000, J. Dermatol 27:434) collagen dress (Olson et al., 1995, Br. J Dermatol 132:587-91) or fabric gauze. The gauze may be secured with Tegaderm (3M, St. Paul, Minn.) (Chen et al., 2000, J. Dermatol 27:434; Olsson et al., 1995, Br J. Dermatol 132:587). Patients are instructed to lie still for a period of time to allow the transplanted cells to adhere to the transplant site.

Claim 1 of 17 Claims

1. A composition for culturing epidermal melanocytes comprising basal medium, serum, one or more antibiotics, one or more growth factors, and one or more natural, physiological cAMP-elevating agents, wherein the one or more natural, physiological cAMP-elevating agents comprises epinephrine, and wherein the one or more growth factors comprises hepatocyte growth factor.

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