Serum-free culture of normal human melanocytes: growth kinetics and growth factor requirements.
Normal human epidermal melanocytes were selectively propagated from mixed (keratinocyte-melanocyte) cultures and primary epidermal cell suspensions in serum-free medium, MCDB 153 containing insulin, bovine pituitary extract (BPE), phorbol-12-myristate-13-acetate (PMA), ethanolamine, phosphoethanolamine, and hydrocortisone. Neonatal foreskin melanocytes (NFMs) replicated more readily than adult melanocytes in culture. Early passage NFMs grown in serum-free medium exhibited a population generation time of 24-48 hours. NFMs assumed a less dendritic appearance and were less pigmented than adult melanocytes. PMA or other protein kinase C-activating phorbol esters significantly enhanced mitogenesis of NFMs; however, cAMP-elevating agents were not required for efficient replication of NFMs. Basic fibroblast growth factor (bFGF) was a potent mitogen for NFMs and replaced the requirement for BPE in the culture medium. NFMs expressed a single class of specific, high-affinity receptors for bFGF, exhibiting a Kd = 3 x 10(-11) M and approximately 76,500 receptors/cell. Neither EGF nor TGF-alpha were mitogenic for NFMs, and TGF-beta reversibly inhibited NFM growth. Rapidly growing, early passage NFMs were shown to have cell cycle times of 19.5, 7.5, and 9 hours for G1, S, and G2/M phases of the cell cycle, respectively. Culture of NFMs to confluence or depletion of growth factors from the culture medium caused reversible, G1 phase-specific, cell cycle growth arrest. Senescence of NFMs was associated with irreversible growth arrest in the G1 phase after 40-45 population doublings in culture. Our data demonstrate that basal medium MCDB 153 can be supplemented with defined factors to cultivate selectively two major constituent cell types of the epidermis, the melanocyte and the keratinocyte.
Pittelkow
M R
MR
Department of Dermatology, Mayo Clinic/Foundation, Rochester, Minnesota 55905.
Shipley
G D
GD
D000328:Adult; D002453:Cell Cycle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D000242:Cyclic AMP / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007231:Infant, Newborn; D007328:Insulin / Q000494:pharmacology; D008544:Melanocytes / Q000166:cytology*; D010703:Phorbol Esters / Q000494:pharmacology; D010902:Pituitary Gland / Q000502:physiology; D011956:Receptors, Cell Surface / Q000502:physiology; D017468:Receptors, Fibroblast Growth Factor; D014020:Tissue Extracts / Q000494:pharmacology; D015291:Transforming Growth Factors / Q000494:pharmacology
D003470:Culture Media; D006133:Growth Substances; D007328:Insulin; D010703:Phorbol Esters; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D014020:Tissue Extracts; D016222:Fibroblast Growth Factor 2; D005346:Fibroblast Growth Factors; D004815:Epidermal Growth Factor; D015291:Transforming Growth Factors; D006493:Heparin; D000242:Cyclic AMP