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M. Valtink, K. Engelmann; Propagation of Human Adult Choroidal Melanocytes in a Serum–Free Culture System . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3353.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To cultivate human adult choroidal melanocytes without serum or toxic or tumor–promoting supplements. Methods: Human choroidal melanocytes were isolated by incubation in dispase II (0.6 U/ml) for 18h after isolation of RPE cells with collagenase. Enzyme reaction was stopped by protease inhibitor, cells were then washed and seeded in serum–free Melanocyte Growth Medium M2 (PromoCell) in uncoated T25 cell culture flasks. Medium was changed 3x/week. If necessary, contaminating RPE cells were eliminated by geneticin treatment for 7d before passaging with trypsin. Trypsinized cells were seeded at 2,000 cells/cm2 on uncoated culture dishes. Eventually, cells were cryopreserved in liquid N2 using medium M2 + 10% DMSO. Purity of the established cultures was checked by immunocytochemical staining. Results: Under serum–free conditions, cultures underwent at least 13.2±3.4 cpd (5.8–18.1 cpd) before they became senescent or were deliberately terminated. Mean generation time was 92.0±18.6 h (56–128 h). After cryopreservation, generation time was markedly decreased but proliferative capacity of the cells was not impaired. Freshly isolated cells were rather large with a bipolar morphology, whereas subcultured cells became smaller with a bipolar to stellate morphology. Confluent cultures consisted of bipolar cells in a whorling pattern and triangular cells in a cobblestone–like pattern. Triangular cells seemed not to be senescent but quiescent, and started to proliferate after trypsinization, showing again a bipolar morphology. Cultured cells lost pigment after initial seeding, but displayed melanogenesis throughout the entire cultivation period. All cultures stained positive for HMB–45 antigen and S–100, and only few cultures contained some single cells that stained positive for RPE specific cytokeratin 8,18. Few cultures showed weak positive staining for MMP–2 or MMP–9. Conclusions: Melanocyte Growth Medium M2 is a commercially available serum–free medium that contains no phorbol esters or supplements like cholera toxin. Using this medium, pure cultures of human adult choroidal melanocytes could successfully be established and maintained for several months.
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