A Comparison Between Phorbol 12 Myristate 13 Acetate and Phorbol 12, 13 Dibutyrate in Human Melanocyte Culture

Abstract

Introduction: Melanocyte culture is an integral part of the studies of skin biology and cosmetic applications. After the introduction of selective medium for the culture of human melanocyte using Phorbol 12-myristate13-acetate (PMA) in 1982, a lot of methods of culturing were tried but till date PMA is a preferred mitogen because of its cost effectiveness compared to growth factors. We have tried to preliminarily evaluate the efficacy of another phorbol ester, Phorbol 12, 13-dibutyrate (PDBu) in melanocyte culture because of its less hydrophobic nature compared to PMA. This property minimizes the trace amount of mitogen in cell culture after washing off and hence does not interfere in other biological assays. Aim: To evaluate the differences in the melanocyte survival rate, morphology and mitotic index when grown in media supplemented with PMA and PDBu. Materials and Methods: Foreskins were collected from children undergoing circumcision. Epidermal cells were isolated from foreskin and cultured using PMA and PDBu. Melanocytes in culture were monitored for the better establishment and documented. In proliferative assay, melanocytes were treated with PMA and PDBu for 24, 48 and 72 hours and proliferation was measured using 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay method. Results: When cultured, melanocytes acquired proliferative status and bipolar morphology quicker in PDBu medium than in PMA medium. Keratinocytes survived as contamination in PMA medium whereas PDBu medium had minimal keratinocytes. MTT assay showed that PDBu has higher proliferative induction capacity than PMA. In even lower concentration of PDBu in medium, melanocytes survived till 72 hours without significant cell loss in compared to PMA medium. Conclusion: PDBu can be a valuable replacement for PMA in human melanocyte culture. Higher proliferation induction, unfavourable to keratinocyte survival and less hydrophobicity make PDBu a promising alternative for quicker establishment of pure human melanocyte cultures especially in cosmetic in vitro experimental dermatology.

Keywords

• cell culture
• melanocyte
• phorbol esters