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


CONCLUSIONS
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.
Tissue Culture Center, New York Eye and Ear Infirmary, NY 10003.
D N
DN
Hu
S A
SA
McCormick
R
R
Ritch
K
K
Pelton-Henrion
D000328:Adult; D000368:Aged; D002455:Cell Division; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D007124:Immunoenzyme Techniques; D008544:Melanocytes / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism; D009418:S100 Proteins / Q000378:metabolism; D014602:Uvea / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure
D003470:Culture Media; D003598:Cytoskeletal Proteins; D005839:Gentamicins; D009418:S100 Proteins; C010680:antibiotic G 418