Investigative Ophthalmology & Visual Science, Vol 34, 2210-2219, Copyright © 1993 by Association for Research in Vision and Ophthalmology

ARTICLES AND REPORTS

Studies of human uveal melanocytes in vitro: isolation, purification and cultivation of human uveal melanocytes

DN Hu, SA McCormick, R Ritch and K Pelton-Henrion
Tissue Culture Center, New York Eye and Ear Infirmary, NY 10003.

PURPOSE. To develop the methods for isolation and cultivation of human uveal melanocytes (UM) from adult donor eyes. METHODS. After removal of the pigment epithelium, the uvea was pretreated in trypsin solution at 4 degrees C overnight, incubated at 37 degrees C with trypsin for 1 hr, then incubated with collagenase for 3 hr. Released cells were collected each hour during the incubation and cultured with F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, isobutylmethylxanthine and cholera toxin. Contaminant cells were eliminated by adding a selective cytotoxic agent, geneticin, when necessary. RESULTS. These methods provide pure melanocyte cultures with high cell yields, good viability, and rapid growth rates. UM isolated and maintained using these methods can be passaged 23 times for a period of 7 mo for more than 35 population doublings. This is comparable to results obtained with cultured neonatal dermal melanocytes and exceeds results obtained with adult dermal melanocytes cultured in media supplemented with phorbol ester, isobutylmethylxanthine, and cholera toxin. CONCLUSION. A method for isolation and cultivation of UM has been developed that yields satisfactory results. Cultured UM may be useful in in vitro studies of UM physiology and may allow development of in vitro models of the pathogenesis of uveal malignant melanoma.

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