A procedure for the isolation of primary cultures of melanocytes from newborn and adult human skin

Abstract

A method to obtain high-density primary cultures of adult and neonatal human melanocytes is described. Keratinocytes and melanocytes are coisolated from split-thickness sections of neonatal foreskin obtained after circumcision, or from adult facial skin removed for cosmetic correction. Cells are released by trypsin and are plated in McCoy's 5A medium containing cholera enterotoxin (1 x 10-9M) and isobutylmethylxanthine (3.3 x 10-5M) to enhance melanocyte and keratinocyte multiplication. Keratinocytes and melanocytes are allowed to grow for 1 week. To remove associated keratinocytes, cells are exposed to 5-fluorouracil (5-FU) (1.92 x 10-5, 3.84 x 10-5, 1.92 x 10-4M). In the presence of low concentrations of 5-FU, keratinocytes are selectively destroyed within 3 weeks, while melanocytes continue to multiply and to form pigment. The differential effect of 5-FU on the multiplication of keratinocytes and melanocytes provides a simple method for obtaining cultures of melanocytes free from other nondendritic cell types present in the epidermis.