Successful culture of adult human melanocytes obtained from normal and vitiligo donors.

Abstract

The development of growth conditions for human melanocytes from uninvolved skin from vitiligo patients and age-matched normal adults is a prerequisite to understanding the etiology of this pigmentary disorder. By using new growth conditions, pure melanocyte cultures were prepared from normal adults and the pigmented skin of vitiligo donors. Both cell types grew without a lag period and were maintained for more than six months (4-8 passages). The cultures could be expanded from several thousand melanocytes in the original cell suspension to several million cells (2-7 x 10(6] in pure culture. To obtain these results, the current standard conditions for the culture of fetal melanocytes were substantially modified. MEM-S medium was less satisfactory than MCDB-153. Adult normal and vitiligo cells also required the presence of exogenous catalase (20 micrograms/ml) during isolation and primary seeding. Thereafter, this enzyme was not necessary. Melanocytes grew best and gave the highest yields if the concentrations of both calcium (200 microM) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) were low (4-8 nM). Other growth factors included in the MCDB-153 media were bFGF, crude bovine pituitary extract, insulin, transferrin, hydrocortisone, 5% FCS, and the antioxidant alpha-tocopherol. Cholera toxin and isobutylmethylxanthine (IBMX) were omitted from the MCDB-153 growth medium because they slowed down growth even at very low concentrations. The results indicate adult and vitiligo melanocytes can be cultured. Preliminary studies of the normal and vitiligo cells indicate that vitiligo melanocytes retain some of the ultrastructural abnormalities observed in skin even when grown in culture.