Establishment of proliferative, pure cultures of pigmented chicken melanocytes from neural tubes

In order to obtain pure cultures of chicken melanocytes, neural tubes were excised from 22-somite stage embryos and placed in culture dishes to allow melanoblasts to migrate out and proliferate. The growth of contaminating cells was inhibited by maintaining the primary cultures in low-calcium and low-magnesium medium supplemented with 32 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). Subsequently the pure cultures of melanocytes were maintained in Ham's F-10 medium supplemented with TPA. The population doubling time was approximately 12 h. The cell density at confluency in medium containing 32 nM TPA, 80 nM TPA, or 32 nM TPA plus 1 nM cholera toxin was 3.4, 5.6, or 8.3 X 10(4) cells/cm2, respectively. The melanocytes were highly pigmented and had tyrosinase activities ranging from 0.7-5.0 mU/mg protein.