SUMMARY 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).
Normal melanocytes from adult human skin are used at the Finsen Laboratory as controls for studies of the effect of drugs and hormones on the growth, pigmentation and motility of melanoma cells. The experiments reported here were performed in an attempt to optimize the separation and cultivation of melanocytes. Keratinocyte cultures obtained simultaneously are used at the Fibiger Laboratory for studies on carcinogenesis and burn healing.The most efficient way of producing a suspension of single, viable cells proved to be that of floating keratomed pieces of skin on trypsin (0.25%, Difco) overnight at 4 0 C or for one hour at 37 0 C. Dermis and epidermis could then easily be separated with tweezers, and alive epidermal cells gently scraped off stratum corneum with a scalpel. A single cell suspension was Dbtained by pipetting.Attempts were made to selectively separate the various epidermal cell types. Gradient centrifugation on a preformed, continuous Percoll gradient produced two layers of cells at densities of approximately 1.100 and 1.050, with the majority of melanocytes in the latter. In an attempt to improve the separati on, ce lIs were centri fuged ina discontinuous Percoll gradient with densities of 1.050 and 1.065. This produced a sediment and two layers of cells that floated on these two respective densities. Although the majority of melanocytes were often found in the second layer of cells and most of the viable keratinocytes usually in the sediment, experience showed this separation technique not to be reliable. An easier and more efficient method pr~ved
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