Serum-free cultivation of adult normal human choroidal melanocytes

Abstract

Cultures of normal choroidal melanocytes are useful in vitro models for the study of melanocyte biology. Current protocols involve the supplementation of culture media with serum, toxins, and phorbol esters, the latter being known as tumour-inducing agents. We therefore sought to establish a protocol to cultivate normal human choroidal melanocytes (NHCMs) without these supplements. NHCMs were isolated by dispase II treatment, after isolation of retinal pigment epithelial (RPE) cells, and seeded in serum-free Melanocyte Growth Medium M2 in uncoated T25 cell culture flasks. Purity of the established cultures was proven by immunocytochemistry. Morphology of the cultured cells was evaluated throughout the entire cultivation period. Eventually, cells were cryopreserved in liquid nitrogen. The cultures underwent at least 11.5 +/- 4.5 doublings before they became senescent or culture was deliberately terminated. Mean generation time was 95.0 +/- 27.7 h. After cryopreservation, generation time was markedly increased, but proliferative capacity of the cells was not impaired. Cultured cells showed bipolar to dendritic morphology; sometimes flattened triangular cells were seen in the cultures. Cultured cells lost pigment after initial seeding but displayed continuous melanogenesis. All cultures stained positive for HMB45 antigen and S-100 and negative for RPE-specific cytokeratins 8, 18. Only few cultures contained single cells that were weakly positive for matrix metalloproteinase (MMP)-2 or MMP-9. The cultivation protocol yields pure cultures of NHCMs that can successfully be maintained for several months without the use of serum, tumour-inducing substances, or toxins.