Culture of normal adult human melanocytes

Melanocytes isolated from normal adult human skin were cultured in vitro. Separation of the epidermis from the dermis by trypsin flotation proved better than collagenase treatment for providing viable cultures of melanocytes with a minimum of fibroblast contamination. Centrifugation on a discontinuous, rather than a continuous Percoll gradient, was more efficient in separating the epidermal cell types. Most of the melanocytes were usually found in one particular layer, and most of the viable keratinocytes were in the sediment. None of the layers produced a uniformly high percentage of melanocytes on routine culture, but enriched melanocyte cultures could be obtained by seeding the epidermal cells in magnesium- and calcium-free medium for 24 to 48 hours, and then transferring them to fibroblast-conditioned medium containing horse serum and polyamines. Melanocytes were identified by their dendritic morphology, ultrastructure, reaction to cholera toxin and pigment production after treatment with melanocyte stimulating hormone. Pure cultures of melanocytes have been cultivated by this method for more than 43 weeks (ten passages).