Abstract: Objective To establish a method for in vitro culture and proliferation of autologous melanocytes,and to observe the therapeutic effect of transplantation of cultured autologous melanocytes on vitiligo.Methods Blisters were induced in vitiligo patients by negative pressure suction.The roof of the blisters was clipped and digested with trypsin,then,the suspension of epidermal cells with melanocytes was selectively cultured and proliferated in serum-free M2 medium.The cells were passaged every week,and after 4-5 passages the purity and biological features of the melanocytes were assessed by immunotluorescence and DOPA staining.Then,the autologous melanocytes were transplanted onto the depigmentation areas of the vitiligo patients.Repigmentation was observed continuously in the transplanted areas.Results Melonacytes could be selectively proliferated in M2 medium.After 4-5 weeks of culture,pure melanocytes were harvested without the contamination of fibroblasts or keratinocytes.Totally,16 vitiligo patients with 28 depigmented areas(size ranging from 2 cm² to 200 cm²) were treated with transplantation of autologous melanocytes.After one month,partial repigmentation was observed in the transplanted areas,and the skin there appeared in light red.After 3-5 months,repigmentation was observed in all the treated areas,while the color of these areas was deeper or lighter than that of the normal skin around.After 6-8 months,the pigmentation leveled off,and these areas were similar in color to the normal skin around.In addition,87.5% lesions experienced more than 50% repigmentation at this time.No scarring or other side effects were observed.Conclusion Transplantation of cultured autologous melanocytes is an effective and safe modality for the treatment of vitiligo,with the advantage of treating large depigmented area with small size of donor epidermis.

Key words: Vitiligo, Transplantation,autologous, Melanocytes