WO2006005977A1

WO2006005977A1 - Novel method for culturing keratinocytes, melanocytes and fibroblasts for skin grafting

Info

Publication number: WO2006005977A1
Application number: PCT/HU2005/000076
Authority: WO
Inventors: Lajos Kemény; Zsuzsa BATA-CSÖRGÖ; Gábor SZABAD
Original assignee: Nagy, Norbert
Priority date: 2004-07-09
Filing date: 2005-07-11
Publication date: 2006-01-19
Also published as: HUP0401385A2; HU227723B1; EP1768716A1; HU0401385D0; WO2006005977B1

Abstract

The present invention in general relates to the field of eukaryotic cell culture and tissue engineering, more closely to a method for the cultivation of eukaryotic cells and the use of cells cultured according to this method for the preparation of cell transplants e.g. autologous cell transplants. In particular, the invention relates to the use of keratinocyte and/or melanocyte and/or fibroblast transplants prepared according to the present invention for the treatment of various dermatological symptoms and diseases. The invention further relates to keratinocyte and/or melanocyte and/or fibroblast transplants, e.g. cell culture preparations, prepared according to the present invention. The invention concerns areas involving eukaryotic cell culture and tissue engineering, particularly fibrin-containing keratinocyte, melanocyte and fibroblast suspensions based on three constituents. The invention is particularly useful in the treatment of various conditions of skin, e.g. chronic ulcers of different origins, burn wounds, post-traumatic wound defects, defects of extensive, benign, naevoid skin changes and of scars after prior dermabrasion, vitiligo, piebaldism, etc.

Description

Novel method for culturing keratinocytes, melanocytes and fibroblasts for skin grafting

The present invention in general relates to the field of eukaryotic cell culture and tissue engineering, more closely to a method for the cultivation of eukaryotic cells and the use of cells cultured according to this method for the preparation of cell transplants e.g. autologous cell transplants. In particular, the invention relates to the use of keratinocyte and/or melanocyte and/or fibroblast transplants prepared according to the present invention for the treatment of various dermatological symptoms and diseases. The invention further relates to keratinocyte and/or melanocyte and/or fibroblast transplants, e.g. cell culture preparations, prepared according to the present invention. Moreover, the present invention is concerned with methods and materials for the initiation of primary cultures of human epidermal keratinocyte and/or melanocyte cells from human tissue, for the storage of these cells in viable frozen condition, for the establishment of secondary cultures recovered from frozen storage which enable serial propagation from the same primary culture, and for the use of these cells in products and procedures for the repair of injury to the skin.

The medical-biotechnological field of tissue engineering encompasses the development, preparation, and modification of implants, biomolecules prepared in vitro, cells or tissues for the substitution or functional assistance of missing or injured body parts and tissues. One focus lies in the preparation of so-called autologous transplants. The underlying principle is to derive cells of the type to be transplanted from a healthy tissue of a patient, the so-called donor area, to culture them in vitro, to use them for the preparation of a transplant, e.g., by introducing them into a suitable carrier matrix, and to finally apply them to an accordingly prepared receptor area of the same patient.

Usually, a small skin specimen is taken from the patient as a full thickness sample from the groin, or as a split thickness graft from the upper leg or arm. Epidermal cell suspension is prepared and plated in vitro. Within two to three weeks, after several passages, a pure keratinocyte culture is established, which is transplanted onto the wound bed. Keratinocytes attach to the wound bed, produce a wide array of growth factors and fasten up the healing process. Since the grafted keratinocytes are autologous, there is no immunological reaction involved in this transplantation technique. The in vitro culturing technique involves subcultivations to expand the number of keratinocytes. However, normal human epidermal keratinocytes have a finite replicative capacity and lifespan. The increased number of cell divisions in vitro leads to telomeric shortening, which in turn reduces the lifespan of the autograft. For this reason it is of outmost importance to use undifferentiated keratinocytes for autologous transplantation purposes. These cells can be found in the basal layer of the epidermis and especially in hair follicles (Alonso L and Fuchs E, Proc Natl Acad Sci 100(suppl 1): 11830-11835, 2003).

A decisive advantage of this kind of autologous therapy lies in that there is usually little danger of immunological responses or infection, which frequently occur in the case of heterologous transplants. In principle, the preparation of such autologous transplants can be imagined on the basis of numerous cell and tissue types, e.g., skin, cartilage, bone, fatty or other kinds of tissue. In practice, the skin is, for example, particularly suitable due to its exposed location and thus easy accessibility. Furthermore, there are various dermatological indicational fields which require-often extended-skin transplantation. In this regard, in particular severe burn injuries as well as badly healing skin ulcers need to be mentioned on the one hand and pigmentation deficiencies, such as vitiligo, on the other. In spite of the evident advantages and promising results the skilled person face many problems in respect of autologous skin transplantation in the art. Cell culture medium

Our knowledge about normal human skin cell biology is mainly based on information acquired by the use of in vitro cultured cells. The standard method for in vitro culture of normal human skin cells includes the use of chemical mitogens, the tumour promoter 12-0-tetradeconyl phorbol-13-acetate (TPA) and the cyclic adenosine 3', 5' monophosphate (cAMP) enhancer, cholera toxin (CT) in most cases, which clearly provides a non-physiological environment for the cells. Thus, a certain need existed in the art to establish a chemical mitogen free skin cell culturing technique. Autologous human serum at 2.5%-5 % was able to provide as good growth support for the cells as the combination of FBS, EGF and BPE. To avoid the use of non- physiological mitogens Nielsen and Donn (Nielsen HL, Don P. Culture of normal adult human melanocytes. Br J Dermatol 1984; 110: 569-80) grew melanocytes in a fibroblast conditioned medium, containing 15% horse serum and polyamines, but melanocytes failed to sustain differentiation. Donatien et. al. (Donatien P, Surleve-Bazeille JE, Thody AJ, Taieb A Growth and differentiation of normal human melanocytes in a TPA- free, cholera toxin-free, low-serum medium and influence of keratinocytes. Arch Dermatol Res 1993; 285: 385-3927) described a system for culturing human melanocytes which avoids the use of non-physiological mitogens. They established melanocyte cultures by co-culturing human epidermal keratinocytes with human epidermal melanocytes, but after the removal of keratinocytes from the culture, melanocytes lost their typical morphological characters. Epidermal stem cells

A crucial step of skin wound healing is epidermal regeneration, i.e., re-epithelialization. Besides interfollicular epidermal keratinocytes from the wound edges, the outer root sheath (ORS) cells from residual hair follicles also contribute to this process (Eisen et al., J Invest Dermatol 15: 145-156, 1955). The ORS of hair follicles is comprised largely of undifferentiated keratinocytes that encompass the cylindrical structures of the hardened inner root sheath and the hair shaft (Montagna and Parakkal, pp. 172-258 in "The Structure and Function of Skin", c. 1974 by Academic Press New York, N. Y.). Recent literature indicates that ORS cells are at a lower level of commitment to differentiation than the basal interfollicular keratinocytes (Coulombe et al., J Cell Biol 109: 2295-2312, 1989; Limat et al., Exp Cell Res 194: 218-227, 1991; Limat et al., Cell Tissue Res 275: 169-176, 1994), and label retaining cells have been detected in the animal as well as the human ORS region which possibly represent stem cells for skin epithelial tissues (Cotsarelis et al., Cell 61: 1329-1337, 1990; Kobayashi et al., Proc Natl Acad Sci USA 90: 7391-7395, 1993; Yang et al., J Invest Dermatol 105: 14-21, 1993; Rochat et al., Cell 76: 1073-1076, 1994; Moll, J Invest Dermatol 105: 14-21, 1995). Human ORS cells isolated from plucked anagen scalp hair follicles can be expanded extensively in vitro (Weterings et al., Brit J Dermatol 104: 1-5, 1981; Limat and Noser, J Invest Dermatol 87: 485-488, 1986; Imcke et al., J Am Acad Dermatol 17: 779-786, 1987; Limat et al., J Invest Dermatol 92: 758-762, 1989). Under conventional submerged culture conditions, ORS cells resemble interfollicular epidermal keratinocytes by both morphologic and biochemical (e.g., keratin profiles) criteria (Stark et al., Differentiation 35: 236-248, 1987; Limat et al., J Invest Dermatol 92: 758-762, 1989; Limat et al., Ann NY Acad Sci 642: 125-147, 1991). In organotypic co cultures with human dermal fibroblasts, i.e., under conditions rnimicking the epidermal environment, ORS cells with respect to histological, immunohistological, ultra structural and biochemical criteria develop a stratified epithelium reminiscent of regenerating epidermis (Lenoir et al., Dev Biol 130: 610-620, 1988; Limat et al, Exp Cell Res 194: 218-227, 1991; Limat et al., Ann NY Acad Sci 642: 125-147, 1991). If such organotypic cultures are grafted onto nude mice, ORS cells form a regular neo-epidermis that is under homeostatic control (Limat et al., Transplantation 59: 1032-1038, 1995). Thus, it was known that human ORS cells are of considerable interest for clinical application.

In the last decade, interest has focused on the use of cultured epithelial cells for wound coverage. First, sheets of cultured autologous interfollicular keratinocytes were grafted successfully on acute wounds, mainly in the treatment of larger third degree burns (O'Connor et al., Lancet 1: 75-78, 1981; Compton et al., Lab Invest 60: 600-612, 1989) but also of epidermolysis bullosa (Carter et al., J Am Acad Dermatol 17: 246-250, 1987), pyoderma gangrenosum (Dean et al., Ann Plast Surg 26: 194-195, 1991; Limova and Mauro, J Dermatol Surg Oncol 20: 833-836, 1994), and wounds after excision of giant congenital nevi (Gallico et al., J Plast Reconstr Surg 84: 1-9, 1989) or separation of conjoined twins (Higgins et al, J R Soc Med: 108-109, 1994). In contrast to the treatment of acute wounds, however, grafting of chronic wounds such as leg ulcers with cultured keratinocytes has been much less successful. Allografts do not result in a permanent take (Fabre, Immunol Lett 29: 161-166, 1991) and thus may be classified as a quite effective but expensive biological dressing (reviewed by Phillips et al., J Am Acad Dermatol 21: 191-199, 1989). A reproducible, major definite take of autologous keratinocyte grafted by various modalities - e.g. (i) sheets of submerged keratinocyte cultures consisting of only a few, noncornified cell layers (Hefton et al., J Am Acad Dermatol 14: 399-405, 1986; Leigh and Purkis, Clin Exp Dermatol 11: 650-652, 1986; Leigh et al., Brit J Dermatol 117: 591-597, 1987; Philips et al., J Am Acad Dermatol 23: 189-198, 1990; Giannotti et al., G Ital Dermatol Venerol 125: 161-167, 1990; Harris et al., Clin Exp Dermatol 18: 417-420, 1993), (ii) trypsinized single cells attached to collagen-coated dressings (Brysk et al., J Am Acad Dermatol 25: 238-244, 1991) or (iii) skin equivalents (MoI et al., J Am Acad Dermatol 24: 77-82, 1991) - has not been convincingly documented. The same holds true for reports on grafting of freshly isolated, autologous interfollicular keratinocytes (Hunyadi et al., J Dermatol Surg Oncol 14: 75-78, 1988) or ORS cells (Moll et al, Hautarzt 46: 548-552, 1995) fixed to the wound bed by a fibrin glue. Disadvantages of bovine serum used during cultivation of the keratinocytes may contribute to reduced take rate, since it resists in keratinocytes (Johnson et al., J Burn Care Rehab 11: 504-509, 1990).

Extensive efforts to cure the outlined problems have been made by several authors. In patent No. US 5968546 (1999) Baur M et al proposed generation of keratinocytes from other root sheath cells for the treatment of skin defects. The authors obtained primary cultures comprising large number of ORS cells explantation of plucked hair follicles on micoporous membranes carrying human fibroblast feeder cells.

Hunziker T and Limat A later taught an improved and simplified method for preparing a keratinocyte culture, wherein the plucked, anagen or growing hairs are explanted and cultured in toto upon microporous membranes carrying human fibroblast feeder cells at their under-surface. In these primary cultures large number of ORS cells can be obtained (international publication WO01/05942 and patents US 6730513 and US 6548058). The subsequent preparation of skin or epidermal equivalents is achieved by the "seeding" of these ORS cells upon a microporous membrane carrying growth-arrested/limited human dermal fibroblast feeder cells. The authors propose the use of a reduced concentration of serum, preferably the use of autologous human serum.

In patent US 6673603 Baetge et al disclose a cell paste comprising keratinocytes and fibroblasts. It is demonstrated by the authors that mosmitotic human fibroblasts and postmitotic keratinocytes secrete growth factors when embedded in fibrin. Moreover, a synergistic effect was observed when fibroblasts and keratinocytes were cultured in combination. Treatment of tissue defects or wounds by administering the cell pastes to a wound site of a patient are also taught.

Contrary to treatment of chronic wounds, vitiligo could be successfully treated by state-of-art methods in many cases.

Olsson M J and Juhlin L review surgical techniques by using epidermal sheet grafts for repigmentation (Olsson M J and Juhlin L Acta Derm Venereol 77: 463-466, 1997). Though results by this method are often satisfactory, in individuals who have large or changing depigmented patches no 100% result can be achieved. The same authors compared later different methods, including transplantation of autologuos melanocytes cultured in M2 medium. In vitiligo vulgaris transplantation of cultured melanocytes resulted in poorer repigmentation than that of epidermal sheets.

Chen et al. (J Am Acad Dermatol 44: 545, 2001) applied an other medium, HU16 medium, comprising among other cholera toxin, have been used for stimulation melanocyte growth. Hu D and McCormick S

(US2005/0064588) improved epidermal melanocyte culture formulations further. Their culture medium comprised a basal medium supplemented with growth factor, agents elevating cAMP levels, but also comprised fetal bovine serum.

It appears that cell culture media of the art suitable for effective growth of melanocytes necessarily comprise either non-physiological chemical substances or fetal bovine serum.

To the best of our knowledge, no authors suggest to use hairy skin, in particular ORS cells in the treatment of vitiligo or similar conditions of leukoderm type.

Moreover, it appears from the above study of the prior art that known methods established for treatment of either acute or chronic wounds by culturing keratinocytes can not be used for treatment of conditions of the skin where melanocytes are to be transplanted, such as leucoderma, including vitiligo and piebaldism; and treatment method for the latter conditions can not be applied in the treatment of wounds. Thus, there is still a need in the art to find improved methods for skin grafting, in particular autologous skin grafting, by cultured cells, in particular by in vitro growth and differentiation of human epidermal keratinocytes and/or melanocytes.

Moreover, there is a need in the art for versatile skin grafting method useful in the treatment of several disease conditions, e.g. both vitiligo and various kinds of wounds. There is also a need to find new donor sites of the skin comprising an increased number of epidermal stems cells.

Furthermore, there is still a need in the art to use advanced cell culture media in autologous skin grafting, in particular media free of chemical mitogens and heterologous serum. BRIEF DESCRIPTION OF THE INVENTION

Autologous skin cell transplantation methods

The invention relates to a method for autologous skin cell transplantation comprising the steps of i) obtaining hairy scalp of a subject having an affected skin area, said hairy scalp comprising, at least partly, the outer root sheath and/or the bulge, ii) obtaining an epidermal cell suspension from the said hairy scalp, iii) separating epidermal cells from other cell types, at least from dermal fibroblasts, and optionally also separating dermal fibroblasts from the hairy scalp, iii) culturing the said epidermal cells in a suitable medium, iv) administering the cultured epidermal cells to the affected skin area.

Epidermal cells can be obtained e.g. by split skin graft of the hairy skin. An advantage of this method is that hair follicle matrix and dermal papilla remain intact, therefore hair can easily grow at the site of biopsy. Preferably, the hairy scalp, e.g. the split skin graft comprises the bulge.

Preferably, said epidermal cells comprise epidermal stem cells. Preferably, the epidermal cells comprise keratinocytes and/or melanocytes.

Preferably in the epidermal cells obtained according to the invention the keratinocytes comprise a higher percentage of K1/K10 negative cells than keratinocytes separated from other skin area, preferably from the groin or from the upper leg. Preferably, the mean value of the ratio of the K1/K10 negative keratinocyte cells is at least 40%, preferably at least 50%. In a preferred method both keratinocytes and melanocytes are used and cultured together in a mixed culture, in a medium providing suitable conditions for both cell types.

Preferably, the culturing medium comprises serum free medium suitable for culturing mammalian cells, preferably lymphocytes and/or keratinocytes. More preferably, the medium used in the invention comprise a serum free lymphocyte medium and a basal medium useful for the preparation of keratinocytes. Preferably, the culturing medium comprises both KBM and Aim-V media.

Preferably, the medium also comprises one or more natural amino acid, preferably any of the following: L-Glutamine, L-Asparagine.

Preferably, the medium also comprises one or more antibiotic and/or an antimycotic agent. It is to be understood that any antibiotic or antimycotic agent effective under the given conditions may be used in the invention.

In a preferred embodiment of any of the above methods both keratinocytes and melanocytes are used and cultured separately in pure cultures.

In a preferred embodiment of any of the above methods keratinocytes and melanocytes are used and cultured in a mixed culture. In a further preferred embodiment the invention relates to a method for autologous skin cell transplantation comprising the steps of i) obtaining epidermal cells from a subject having an affected skin area, said epidermal cells comprising both keratinocytes and melanocytes ii) isolation of said epidermal cells from other cell types, at least from dermal fibroblasts and separation of keratinocytes and melanocytes, iii) culturing the said keratinocytes and melanocytes separately in suitable media, iv) administering the cultured keratinocytes and/or melanocytes in defined ratios to the affected skin area.

These cultures can be reconstituted later in appropriate or desired ratios, e.g. as required by the given condition of the affected skin area to be treated. For examples, in conditions, wherein the wound is deep, as a general rale more fibroblasts are needed, at surface injuries or scars, the ratio of keratinocytes should be higher, whereas at vitiligo, the ratio of melanocytes is to be relatively increased.

Keratinocytes and melanocytes can be separated during the isolation process based on a difference in the time of release. Preferably, isolation is carried out by trypsinization. In a method of the invention fibroblasts are separated and cultured separately, and preferably at least one of the following steps is carried out: a) supernatant of the fibroblast culture or a portion thereof is added to the epidermal cell culture or the culture of keratinocytes and/or melanocytes, b) cultured fibroblasts are administered to the affected skin area. The supernatant of the fibroblast culture is termed as Fibroblast Conditioned Medium (FCM), FCM used in the method of the invention can be any fibroblast culture supernatant as defined herein.

In a preferred method the cultures of keratinocytes, melanocytes and fibroblast are stored, e.g. frozen and used at a later time. Portions thereof can be used even several times in repeated treatments.

The affected skin area can be e.g. any of the following: chronic ulcers of different origins, burn wounds, post-traumatic wound defects, covering defects of extensive, benign, naevoid skin changes, scars after prior dermabrasion, vitiligo etc.

Manufacture of a cultured cell preparation

According to a further aspect the invention relates to a method for the manufacture of a cultured cell preparation for skin cell transplantation, preferably autologous skin cell transplantation, comprising the steps of i) obtaining hairy scalp of a subject, said hairy scalp comprising, at least partly, the outer root sheath and/or the bulge, ii) obtaining epidermal cells from the said hairy scalp iii) separating epidermal cells from other cell types of the hairy scalp, at least from dermal fibroblasts, and optionally also separating dermal fibroblasts from the hairy scalp, iv) culturing the said epidermal cells in a suitable medium, thereby obtaining a cell suspension, v) formulating said cell suspension to a cultured cell preparation for skin cell transplantation to an affected skin area.

Preferably, said epidermal cells comprise epidermal stem cells. Preferably, epidermal cells comprise keratinocytes and/or melanocytes.

In a preferred embodiment, both keratinocytes and melanocytes are used and cultured together in a mixed culture, in a medium providing suitable conditions for both cell types. Preferably, the culturing medium used in this method is a medium as defined herein, e.g. in the "Cell culture media" section below or in the Examples. In a preferred embodiment, both keratinocytes and melanocytes are used and cultured separately in pure cultures.

In a further preferred embodiment the invention relates to a method for the manufacture of a cultured cell preparation for autologous skin cell transplantation comprising the steps of i) isolation of epidermal cells, obtained from hairy scalp of a subject, from other cell types, at least from dermal fibroblasts, said hairy scalp comprising, at least partly, the outer root sheath and/or the bulge, said epidermal cells comprising both keratinocytes and melanocytes ii) isolation of said epidermal cells from other cell types, at least from dermal fibroblasts and separation of keratinocytes and melanocytes, iii) culruring the said keratinocytes and melanocytes separately in suitable media, iv) formulating said cell suspension to a cultured cell preparation for skin cell transplantation.

These cultures can be reconstituted later in appropriate, e.g. any pre-determined or desired ratios, e.g. as required by the given condition to be treated, as explained above. Keratinocytes and melanocytes can be separated e.g. during the isolation process based on a difference in the time of release.

In a method fibroblasts are separated and cultured separately, and preferably at least one of the following steps can be carried out: a) supernatant of the fibroblast culture or a portion thereof is added to the epidermal cell culture or the culture of keratinocytes and/or melanocytes, b) cultured fibroblasts are formulated to a cultured cell preparation for skin cell transplantation. Optionally, the cultured fibroblasts are added to the melanocyte and/or keratinocyte culture(s).

The supernatant of the fibroblast culture is termed as Fibroblast Conditioned Medium (FCM). Thus, the invention relates to a method for the preparation of a Fibroblast Conditioned Medium, comprising the steps of: i) obtaining hairy scalp of a subject having an affected skin area, said hairy scalp comprising, at least partly, the outer root sheath and/or the bulge, ii) separating dermal fibroblasts, iii) culturing the said dermal fibroblasts separately in a suitable medium, iv) supernatant of the fibroblast culture or a portion thereof is obtained.

Preferably, dermal fibroblasts are separated from dermis by effecting release of epidermal cells, preparing a dermal cell suspension and seeding it in an appropriate culture medium.

Preferably, the supernatant of the fibroblast culture (FCM) is collected after the fibroblast cultures reached an at least 50%, 60%, 70% or preferably an at least 80% or, highly preferably, at least 90% confluency.

Preferably, the dermal fibroblasts are cultured in any culture medium as defined herein. In a preferred embodiment, primary cultures of keratinocytes, melanocytes and/or fibroblasts can be stored, e.g. in a frozen state, and secondary cultures can later be recovered from frozen storage which enable serial propagation from the same primary culture. Cultured cell preparations

In a further aspect the invention relates to a cultured cell preparation for autologous skin cell transplantation in a subject comprising cultured melanocytes and/or keratinocytes cultured from cells taken from said subject, said preparation being obtainable by any of the methods of the invention; and a cultured cell preparation for autologous skin cell transplantation in a subject comprising cultured fibroblasts cultured from cells taken from said subject, said preparation being obtainable by any of the methods of the invention.

The cultured cell preparations of the invention can be stored, e.g. in a frozen state, for a longer period and used later or several times. They can also be used in other recipients, provided that no rejection of the grafted tissue occurs.

In a still further aspect the invention relates to cultured cell preparation kit for autologous skin cell transplantation in a subject comprising in separate vials cultured melanocytes, keratinocytes and fibroblasts originally obtained from said subject prepared by any of the methods of the invention, useful for use in a ratio required by the condition of the affected skin area to be treated. Cell culture media

Preferably, the culture medium used in the invention comprises, as major component, a serum free medium suitable for culturing mammalian cells, preferably lymphocytes and/or keratinocytes. More preferably, the medium used in the invention comprise as major components a serum free lymphocyte medium and a basal medium useful for the propagation of keratinocytes (keratinocyte basal medium). The serum free lymphocyte medium can be any medium commercially available for that purpose or serum free media expectably suitable to culture lymphocites.

The keratinocyte basal medium can be any medium commercially available for that purpose or other basal media known to be suitable to culture keratinocytes.

The two types of media as components may be used actually in any ratios. Preferably, any of them is present at least about 10%, 20%, 30% or 40%. Highly preferably, their ratios are approximately equal. Preferably, the culturing medium comprises both KBM and Aim-V media.

It is to be understood, however, that any medium described in the art for culturing keratinocytes, melanocytes and/or fibroblasts may be used in the present invention. However, the present inventors think that use of media described herein represent a preferred embodiment. Preferably, the medium also comprises one or more natural amino acid, preferably any of the following:

L-Glutamine, L-Asparagine.

Preferably, the medium also comprises one or more antibiotic and/or an antimycotic agent. It is to be understood that any antibiotic or antimycotic agent effective under the given conditions may be used in the invention. In a preferred embodiment, any of the media used for culturing the cells comprises autologous serum.

The concentration of the autologous serum is preferably less than 10%, 5%, 4% or highly preferably, less than 3%, e.g. about 2 to 3%, e.g. 2,5%.

The medium of the invention may comprise FBS (foetal bovine serum). However, preferably the medium does not comprise FBS. The medium of the invention may comprise a growth factor, e.g. any of the following: EGF (epidermal growth factor), BPE (bovine pituitary extract), KGF (keratinocyte growth factor) or FGF (fibroblast growth factor) etc. In a highly preferred embodiment the culture media lacks any growth factors and any mitogens.

In an aspect, the invention relates to a FCM obtainable by the method of the invention or any cell culture medium comprising FCM of the invention.

The FCM of the invention is a medium which is a supernatant of dermal fibroblast cells obtained from hairy scalp of a subject, said hairy scalp comprising, at least partly, the outer root sheath and/or the bulge.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Analyses of freshly isolated keratinocytes from hairy scalp skin showed that the percentage of

K1/K10 negative cells was significantly higher (52.63 ± 10.21 %) than in the case of keratinocytes separated from groin (35.37 ± 4.47 %) or from the upper leg (36.11 ± 3.24 %) (Fig. 1 A-C). Moreover hairy scalp yielded a higher number of epidermal cells from the same size of skin specimen (Fig. 1 D).

Figure 2. MTT assays to determine the effect of systematic removal of various growth factors from cell mitogen free culture media (50% AIM-V and 50% KBM)

Figure 2a: bar A the medium comprised EGF, BPE and FBS; bar B: Removal of EGF; bar C: removal of BPE; bar D: removal of both EGF and BPE; bar E: removal of only FBS from the media; bar F: no supplement added (no EGF, no BPE, no FBS),

Figure 2b MTT assays to determine the optimal FBS concentration for cell growth in the presence of EGF and BPE.

Figure 3. MTT assays to determine the effect of systematic removal of various growth factors from cell mitogen free culture media (50% AIM-V and 50% KBM plus autologous serum) bar H: Cell growth in the media supplemented with EGF, BPE and 2.5% autologous serum instead of FBS; bar I: lack of EGF; bar J: lack of BPE (Table 1. J); bar G: media containing AIM-V and Keratinocyte- Basal Media (v:v) supplemented with only 2.5% autologous serum and without any supplements (no EGF, no BPE,); bar A: media containing EGF, BPE and FBS

Figure 4. Separation of melanocytes from keratinocytes

Figure 5. Pure cultures of melanocytes, obtained by separation from keratinocytes during trypsinization Figure 6. The course of healing after autologous keratinocyte transplantation in chronic leg ulcer. On the same cms medial ulcer (left column, "a-d") was transplanted, while lateral ulcer (right column, "e-h") was non-treated. Both ulcers received the same supportive therapy.

"a" - clinical status prior operation; "b" and "f - treated and non-treated ulcer 7 weeks after transplantation; "c" and "g" - treated and non-treated ulcer 10 weeks after transplantation; "d" -healed ulcer 13 weeks after only one autologous keratinocyte transplantation; "h" - non-treated ulcer after 16 weeks; "e" - relation of treated and control ulcer on the same eras.

Figure 7a Preparation of hairy scalp from a patient.

Figure 7b Light microscopic image of a keratinocyte culture. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of autologous keratinocyte and/or melanocyte and/or fibroblast transplants prepared according to the present invention for the treatment of various dermatological symptoms and diseases. The present inventors recognized that it is advantageous to use hairy scalp of a subject as a donor area for obtaining cells in skin transplantation. It is particularly useful to obtain a hairy scalp comprising the bulge.

As demonstrated herein the bulge is a rich source of keratinocyte stem cells.

Keratins are type I and II intermediate filament proteins which form a cytoskeletal network within all epithelial cells. They are expressed in pairs, in a differentiation specific fashion. Kl and KlO are early differentiation markers of human keratinocytes, which are expressed in the suprabasal layers of the epidermis. Expression of Kl and KlO keratins mirrors the commitment to differentiation and is among the earliest events in the program of cellular terminal differentiation. K1/K10 negative keratinocytes represent undifferentiated cells with high proliferative potential and high regenerative, capacity that is especially important in wound healing. Thus the proportion of K1/K10 negative cells in transplanted keratinocytes is important both for the short term and long term clinical outcome.

Moreover, by obtaining hairy scalps melanocytes can also be obtained in the same sampling step. Since melanocytes can be cultured similarly to keratinocytes, the problem of pigmentation of the skin graft can be solved. The keratinocytes and the melanocytes may be cultured in mixed cultures; however, after separation, these cells can be cultured separately as well, and admixed later in desired rations. This enables the application of the method in various conditions from chronic wounds to leukoderm conditions, e.g. vitiligo.

The present invention also facilitates the initiation of primary cultures of human epidermal keratinocyte cells from human tissue, the storage of these cells in viable frozen condition, the establishment of secondary cultures recovered from frozen storage which enable serial propagation from the same primary culture, and subsequent use of these cells in products and procedures for the repair of injury to the skin.

Though in a preferred embodiment of the present invention autologous transplants are used to avoid immunological rejection of foreign tissue, it is to be understood that allogenic cell cultures may be used as well, provided that said cells are obtained from donors who are genetically related to the recipient and share the same transplantation antigens on the surface of their respective cells (melanocytes, keratinocytes or fibroblasts). Alternatively, if a related donor is unavailable, antigenetically matched donors should be sought. It is to be noted here that melanocytes do not express HLA antigen, thus, allogenic melanocyte transplantation is possible (see US2005/0064588).

Since from the above-mentioned donor site also fibroblasts can be obtained, according to the invention fibroblast cultures can be prepared and added to the cultures of keratinocytes and melanocytes in a desired ratio. The fibroblasts can be used to obtain a fibroblast conditioned medium useful in culturing the other cell types.

Moreover, according to the invention novel serum free media, in particular FBS free media are provided for culturing, producing and maintaining normal or continuous keratinocytes and/or melanocytes and/or fibroblasts in tissue culture. These novel media are also useful for isolating, establishing human skin cells for obtaining continuous keratinocytes, melanocytes and fibroblasts according to the invention. The invention allows to provide primary keratinocytes or melanocytes or fibroblasts produced under serum free conditions without the use of any feeder cells, wherein said primary keratinocytes and melanocytes and fibroblasts are useful in skin grafting. Thereby the invention also minimizes the risk of disease transmission, e.g., by clinical use of blood products, and by using autologous serum or avoiding the use of serum. Transplantation of the cultured cells can be carried out by any method available for this purpose. Such methods are taught eg. in international publication WO01/05942 and patents US 6730513 and US 6548058, in patent US 6673603, and documents referred therein. Also, as a fibrin glue, any type of appropriate fibrin glue can be applied. The choice of the appropriate method is well within the skilles of a person skilled in the art.

EXAMPLE 1

Separation of Keratinocvte stem cells

Biopsies were taken from three patients after approval of the biopsy and transplantation protocol by the Ethical Committee of the University of Szeged. AU patients were informed of the procedures and all have signed the consent form. The samples were taken from three different sites under local anesthesia: split skin graft of the hairy skin, full thickness graft of the groin and split thickness graft of the upper leg. All samples were 0,25 mm thick and 9cm² in size. Each individual (ages 61-74) had chronic leg ulcers for over 10 years. Skin specimens were first washed in an isotonic solution supplemented with antibiotic, antimycotic solution (Sigma Laboratories). Subcutis and part of the dermis was removed by mechanical means. Overnight incubation in dispase solution (Grade II, Roche Molecular Biochemicals) was carried out at 4 °C to separate the dermis from the epidermis. Next day the skin specimen was further incubated in dispase at 37 ⁰C for 2 hours, than the epidermis was peeled of the remaining dermis. The resulting epidermis was incubated in 0.25 %(w/v) trypsin (Gibco Laboratories) for 30 minutes at 37 °C. Following trypsinization, the epidermis was mechanically torn apart and vigorously washed to release epidermal cells. The epidermal cell suspension was filtered through a 100 mm nylon mesh (BioDesign Inc.) and centrifuged at 1100 rpm for 10 minutes at 4 °C. K1/K10 negative keratinocytes were detected in freshly separated epidermal keratinocytes from hairy scalp, groin and upper leg skin biopsies by flow cytometric analyses using a previously described method (Bata-Csorgo Zs., Hammerberg C, Voorhees J. J., Cooper K. D. Flow cytometric identification of proliferative subpopulations within normal human epidermis and the localization of the primary hyperproliferative population in psoriasis. J Exp Med. 1993 1;178(4):1271-81.).

Analyses of freshly isolated keratinocytes from hairy scalp skin showed that the percentage of K1/K10 negative cells was significantly higher (52.63 ± 10.21 %) than in the case of keratinocytes separated from groin (35.37 ± 4.47 %) or from the upper leg (36.11 ± 3.24 %) (Fig. 1 A-C). Moreover hairy scalp yielded a higher number of epidermal cells from the same size of skin specimen (Fig. 1 D).

EXAMPLE 2

General method for separation of epidermal cells:

The basic epidermal cell-culture media consisted of: AIM-V serum free lymphocyte medium and Keratinocyte-Basal Medium (both from Gibco Laboratories), v:v, supplemented with L-Glutamine and antibiotic/antimycotic solution containing penicillin, streptomycin and amphotericin B (Sigma Laboratories). CeIl separation

Skin specimens were first washed in an isotonic solution supplemented with antibiotic, antimycotic solution (Sigma Laboratories). Subcutis and part of the dermis was removed by mechanical means. Overnight incubation in dispase solution (Grade II, Roche Molecular Biochemicals) was carried out at 4 ⁰C to separate the dermis from the epidermis. Next day the skin specimen was further incubated in dispase at 37 ⁰C for 2 hours, than the epidermis was peeled of the remaining dermis. The resulting epidermis was incubated in 0.25 %(w/v) trypsin (Gibco Laboratories) for 30 minutes at 37 °C. Following trypsinization, the epidermis was mechanically torn apart and vigorously washed to release epidermal cells. The epidermal cell suspension was filtered through a 100 mm nylon mesh (BioDesign Inc.) and centrifuged at 1100 rpm for 10 minutes at 4 ⁰C. The resulting epidermal cell suspension was seeded in culture media on 75 cm2 tissue culture plastic dishes (Corning Costar Corporation) at a cell density of 5x10⁶ cells/cm² in 15 ml culture media. Cells were washed with fresh culture media 12-18 hours after seeding to remove floating cells from the culture. Cultivation of the cells up to subconfluency (37 deg. C, 5% CO2, change of the medium when necessary, as a rule every second or third day). Cultures reached ~90% confluency at an average time of 7 - 9 days. Confluent primary cultures were submitted to PBS (phosphate buffered saline) with 0.05% EDTA and were harvested by trypsinization with 0.01 %(w/v) trypsin (Gibco Laboratories). Cell yield was divided equally into two parts.

To determine the role of the different growth factors in the chemical mitogen free media (Table 1. A), a series of MTT assays were carried out with different culture media (Table 1. B-F). MTT assays can be carried out as follows (Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65 (1-2): 55-63). Melanocytes were seeded in 96 well culture plates at a density of 5xlO³ cells/well and grown in different types of culturing media for 72 hours. Dimethyl tiazol tetrazolium solution (MTT solution) 10 μl 5 mg/ml in PBS was added to each well. After incubation for 3 hours at 37 ⁰C, the medium was gently removed from each well and 100 μl dimethyl sulfoxide was added. After 30 minutes, the intensity of the colour reaction was determined by spectrophotometer at 550 nm and a reference wavelength of 650 nm.

Removal of EGF from the media (Table 1. B) resulted in a 14% decrease of cell number. (Fig. 2a, bar B). Without BPE (Table 1. C) cell growth decreased by 22%. (Fig. 2a, bar C) In the media that lacked both EGF and BPE (Table 1. D) the cell number decreased by 25% (Fig. 2a, bar D), Leaving out only FBS from the media (Table 1. E) resulted in a 31% decrease in growth (Fig. 3 a, bar E). If there were no supplements added (no EGF, no BPE, no FBS, Table 1. F), cell growth decreased by 38% (Fig. 2a, bar F).

In a separate set of experiments MTT assays were performed to determine the optimal FBS concentration for cell growth in the presence of EGF and BPE. Although cells grew without FBS, higher percentages of FBS (from 7% to 10%) resulted in a significant increase in growth (Fig. 2b). Next, we leave out animal derived substances from the culture media. FBS was changed to autologous human serum in these cultures (Table 1. G,H,I,J). Cell growth in the media that was supplemented with EGF, BPE and 2.5% autologous serum instead of FBS, showed a significant increase (Fig. 3 bar H). We again systematically left out supplements from the culture media. The lack of EGF (Table 1. 1) resulted in a slight decrease of growth rate (Fig. 3, bar I), while the lack of BPE (Table 1. J) showed a marked decrease in growth rate (Fig. 3, bar J). The media containing AIM-V and Keratinocyte-Basal Media (v:v) supplemented with only 2.5% autologous serum and without any supplements (no EGF, no BPE) resulted in a growth rate equal to the media containing EGF, BPE and FBS (Table 1. G and A, Fig. 3, bars G and A).

Serum separation

Plastic surgery patients have volunteered to provide blood for the experiments. Informed consent approved by Institutional Review Board was obtained from all volunteers. Blood sampling took place according to the standard techniques for vacutainer tubes. Centrifuging took place 30 minutes after sampling for 15 minutes at 1300 rpm. After correct centrifugation the clear serum above the gel separator was collected.

Table 1.

The composition of the different culture media tested for human epidermal melanocyte growth.

AIM-V serum free media (AIM-V); Keratinocyte serum free media (KBM); epidermal growth factor (EGF) = 5 ng/ml; bovine pituitary extract (BPE) = 50μg/ml; foetal bovine serum (FBS) = 2.5%; autologous serum (AS) = 2.5%

EXAMPLE 3

Cultivation of dermal fibroblasts

Human Autolog Serum (HAS) has a stimulatory effect on the growth rate of dermal fibroblasts. The minimal stimulatory concentration of HAS was determined by MTT assay. Skin specimens were first washed in an isotonic solution supplemented with antibiotic, antimycotic solution (Sigma Laboratories). Subcutis and part of the dermis was removed by mechanical means. Overnight incubation in dispase solution (Grade II, Roche Molecular Biochemicals) was carried out at 4 ⁰C to separate the dermis from the epidermis. Next day the skin specimen was further incubated in dispase at 37 ⁰C for 2 hours, than the epidermis was peeled of the remaining dermis. The resulting dermis was mechanically thorn apart and incubated in enzymatic solution (9,8 ml RPMI + 100 μl lmM-os Na piruvate + 100 μl Hepes + 1 mg DNAse + 27 mg Collagenase + 12,5 mg Hyalorunidase) for 3 hours at 37°C. Following enzymatic digestion, the dermis was vigorously washed to release epidermal cells. The dermal cell suspension was filtered through a 100 mm nylon mesh (BioDesign Inc.) and centrifuged at 1100 rpm for 10 minutes at 4 ⁰C. The resulting dermal cell suspension was seeded in culture media on 75 cm2 tissue culture plastic dishes (Corning Costar Corporation) at a cell density of 5x10⁶ cells/cm² in 15 ml culture media containing 5% autolog serum. Higher percentages of HAS (from 7% to 10%) did not result significant increase in clonal growth. Cells were washed with fresh culture media 12-18 hours after seeding to remove floating cells from the culture. Further cultivation of fibroblast takes place in the same medium but with 2.5 % autolog serum. More than 2.5% HAS (5%, 7%, 10%) in the medium did not support superior growth after the attachment of cells. Cultivation of the cells up to subconfluency (37 deg. C, 5% CO2, change of the medium when necessary, as a rule every second or third day). From the fifth day of cultivation the supernatant of the fibroblast culture was collected and used as supplement for epidermal cell cultures in appropriate concentration, or frozen at -20 ⁰C until further use. Cultures reached ~90% confluency at an average time of 7 - 9 days. Confluent primary cultures were submitted to PBS with 0.05% EDTA and were harvested by trypsinization with 0.01 %(w/v) trypsin (Gibco Laboratories). Cell yield was divided equally into two parts and subcultured separately.

EXAMPLE 4 Characterization of keratinocvtes and melanocytes growth in medium containing fibroblast conditioned medium

Primary cell cultures of keratinocytes and melanocytes were cultivated in medium consisted of: AIM-V serum free lymphocyte medium and Keratinocyte-Basal Medium (both from Gibco Laboratories), v:v, supplemented with L-Glutamine and antibiotic/antimycotic solution containing penicillin, streptomycin and amphotericin B (Sigma Laboratories) as described in Example 2 supplemented with 2,5 % human autologous serum and various amount of fibroblast conditioned medium. Fibroblasts were cultivated as described in Example 3. From the fifth day of cultivation cultures reached ~90% confluency and the supernatant of the fibroblast culture was collected and termed as Fibroblast Conditioned Medium (FCM). MTT assays were performed to determine the optimal FCM concentration for cell growth in the presence of 2.5 % human serum and 0-25% FCM. The highest proliferation rate was observed in the presence of 10% FCM. Increasing amount of FCM 15%, 20%and 25% did not result superior proliferation. For further cultivations we used 10% FCM supplemented medium.

EXAMPLE 5 Autologous Keratinocytes and Melanocytes mixed culture

The basic epidermal cell-culture media consisted of: AIM-V serum free lymphocyte medium and

Keratinocyte-Basal Medium (both from Gibco Laboratories), v:v, supplemented with L-Glutamine and antibiotic/antimycotic solution containing penicillin, streptomycin and amphotericin B (Sigma Laboratories).

Epidermal cell cells were separated as described in Example 1. This cell suspension contains mainly keratinocytes and melanocytes. 5xlO⁶ cells were seeded into a 72 cm² flask (Corning) in KBM:Aim-V (1:1) medium supplemented with 1 %(w/v) L-glutamine solution (SIGMA), 1% AB-AM solution (SIGMA) and 5 % autolog serum. Cells were washed with fresh culture media containing 2,5% autolog serum, 1 %(w/v) L- glutamine solution (SIGMA) and 1% AB-AM solution (SIGMA) 12-18 hours after seeding to remove floating cells from the culture. Cultivation of the cells up to subconfluency (37 deg. C, 5% CO2, change of the medium when necessary, as a rule every second or third day). Cultures reached ~90% confluency at an average time of 7 — 9 days. From the sixth day of cultivation the medium is also supplemented with fibroblast conditioned medium in 10% final concentration. Confluent primary cultures were submitted to PBS with 0.05% EDTA and were harvested by trypsinization with 0.01 %(w/v) trypsin (Gibco Laboratories). Cell yield was divided equally into two parts and cultivated in media containing 2,5% autolog serum, 1 %(w/v) L- glutamine solution (SIGMA), 1% AB-AM solution (SIGMA) and 10% fibroblast conditioned medium.

EXAMPLE 6

Separation of pure cultures of keratinocytes and melanocytes from mixed culture

Skin biopsy was prepared according to the general method in example 5. Primary culture of epidermal cells, preferably keratinocytes and melanocytes was cultivated in medium (KBM+AIMV+Lglu+ABAM+2,5 % has+10% FKM). After the 2^nd passage the melanocytes and keratinocytes were separated upon differential trypsination. To establish pure melanocyte cultures, keratinocytes were progressively removed from the primary culture through the first two passages. Melanocyte attachment to the culture disk differs from the attachment of keratinocytes. During trypsinization melanocytes release 2-3 minutes earlier than keratinocytes, thus enabling separation of melanocytes from keratinocytes (Fig. 4). With this simple separation technique pure cultures of melanocytes were gained (Fig. 5). Cultures were grown at 37 ⁰C in humidified atmosphere containing 5 % CO₂. With this method we have routinely grown human epidermal melanocytes from several human skin specimens. Cells have been cultured through more than 8-9 passages and have shown their typical characters in both low and high passage numbers. From a 9-12 cm² biopsy with this method enough cells could be produced to cover Im² surface. The epidermal cells cultured this way could be cryopreserved and reconstituted at a later time.

EXAMPLE 7

Keratinocyte cultures have been established and expanded as described in Example 6. Only 2 weeks of culturing was necessary to achieve the suitable cell number for transplantation (3xlO⁶ / 100cm²). The patients were transplanted with autologous keratinocytes using the Tissucol system from Baxter. Clinical pictures of one of our representative patient is shown (Fig. 6). Our 74 year old female patient had leg ulcer for 15 years on both the lateral and medial side of her left cms. We performed autologous keratinocyte transplantation in only one occasion on the medial side. Planimetry was performed on both sides. Both ulcers received the same supportive local therapy. While the medial ulcered healed in just 96 days, the lateral showed no significant improvement in size.

In summary, it can thus be stated that skin cell transplantations in connection with the methods and compositions according to the present invention disclosed herein not only much simpler to carry out as compared to other methods, but in addition also significantly promote the success of the transplantation or the repigmentation in question. This aspect is in particular important for example in the context of vitiligo therapy.

Finally, it was also found that autologous skin cell transplants comprising cultured autologous keratinocytes and/or melanocytes and/or fibroblasts in a carrier matrix exhibiting plasticity and a gel-like consistency also exhibit very good transport and application properties.

Thus, the inventive keratinocyte, melanocyte and fibroblast suspensions are useful for the in situ production of (cell) transplants, e.g. autologous (cell) transplants. The invention particularly relates to the use of transplants for treating patients with chronic ulcers of different origins, with burn wounds, post-traumatic wound defects, and for covering defects of extensive, benign, naevoid skin changes and of scars after prior dermabrasion, vitiligo, piebaldism, etc. In chronic leg ulcers and extensive two and three degree burns autologous keratinocyte transplantation is a new line of treatment instead of conventional split thickness grafts. In such cases autologous keratinocytes are prepared and cultured from the patient's own skin. Donor site for such skin samples is usually the groin, the upper leg and arm. Since hair follicles contain a higher number of undifferentiated keratinocytes, keratinocyte cultures established from hairy scalp have a better mitotic potential, making them more suitable for keratinocyte transplantation. The invention allows to produce transplants, which contain keratinocytes or melanocytes or fibroblasts, production of the keratinocyte composition or melanocyte composition or fibroblast composition, and to produce collagen-free two-constituent compositions.

Claims

1 Method for autologous skin cell transplantation comprising the steps of i) obtaining hairy scalp of a subject having an affected skin area, said hairy scalp comprising, at least partly, the outer root sheath and/or the bulge, ii) obtaining an epidermal cell suspension from the said hairy scalp, iii) separating epidermal cells from other cell types of the hairy scalp, at least from dermal fibroblasts, and optionally also separating dermal fibroblasts from the hairy scalp, iii) culturing the said epidermal cells in a suitable medium, iv) administering the cultured epidermal cells to the affected skin area.

2. The method of claim 1 wherein epidermal cells comprise keratinocytes and/or melanocytes.

3. The method of claim 2 wherein keratinocytes and melanocytes are separated from each other and cultured separately in pure cultures.

4. The method of claim 3 wherein the keratinocyte and melanocyte cultures are reconstituted later in desired ratios.

5. The method of any of claims 1 to 4 wherein fibroblasts are also separated from the hairy scalp and cultured separately, and at least one of the following steps is carried out: a) supernatant of the fibroblast culture or a portion thereof is added to the epidermal cell culture or the culture of keratinocytes and/or melanocytes, b) cultured fibroblasts are administered to the affected skin area, in a desired ratio relative to the other cells.

6. Method for the manufacture of a cultured cell preparation for skin cell transplantation comprising the steps of i) obtaining hairy scalp of a subject, said hairy scalp comprising, at least partly, the outer root sheath and/or the bulge, ii) obtaining epidermal cells from the said hairy scalp iii) separating epidermal cells from other cell types of the hairy scalp, at least from dermal fibroblasts, and optionally also separating dermal fibroblasts from the hairy scalp, iv) culturing the said epidermal cells in a suitable medium, thereby obtaining a cell suspension, v) formulating said cell suspension to a cultured cell preparation for skin cell transplantation to an affected skin area.

7. The method of claim 6 wherein epidermal cells comprise keratinocytes and/or melanocytes.

8. The method of claim 7 wherein both keratinocytes and melanocytes are used and cultured together in a mixed culture, in a medium providing suitable conditions for both cell types.

9 The method of claim 7 wherein keratinocytes and melanocytes are separated from each other and cultured separately.

10 The method of claim 9 wherein keratinocyte and melanocyte cultures are reconstituted in a pre¬ determined ratio to obtain a cell suspension comprising a desired ratio of both keratinocytes and melanocytes and this cell suspension is formulated to a cultured cell preparation for skin cell transplantation.

11. The method of any of claims 6 to 10 wherein fibroblasts are also separated from the hairy scalp and cultured separately, and at least one of the following steps is carried out: a) supernatant of the fibroblast culture or a portion thereof is added to the epidermal cell culture or the culture of keratinocytes and/or melanocytes, b) cultured fibroblasts are formulated to a cultured cell preparation for skin cell transplantation and optionally added to keratinocyte and melanocyte culture(s) in a pre-determined ratio.

12. A cultured cell preparation or a cultured cell preparation kit for skin cell transplantation in a subject, said cell preparation or kit comprising cultured melanocytes and/or keratinocytes and/or fibroblasts and being obtainable by a method of any of claims 6 to 11.

13. Method for the preparation of a fibroblast conditioned medium, comprising the steps of: i) obtaining hairy scalp of a subject having an affected skin area, said hairy scalp comprising, at least partly, the outer root sheath and/or the bulge, ii) separating dermal fibroblasts from the hairy scalp, iii) culturing the said dermal fibroblasts separately in a suitable medium, iv) supernatant of the fibroblast culture or a portion thereof is obtained.

14. A cell culture medium for culturing keratinocytes and/or melanocytes, said medium comprising fibroblast conditioned medium obtainable by the method of claim 13.