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D.–N. Hu, S.A. McCormick; Isolation, Cultivation and in vitro Study of Conjunctival Melanocytes With Comparison to Epidermal and Uveal Melanocytes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4946.
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© ARVO (1962-2015); The Authors (2016-present)
To development the methods for isolation and cultivation of conjunctival melanocytes and to compare these cells with epidermal and uveal melanocytes in vitro.
The conjunctiva was micro–dissected from donor eyes and digested with trypsin. Cells released were cultured with HU16 medium (F12 medium with serum, basic fibroblast growth factor, isobutylmethylxanthine and cholera toxin). After 5–7 days, geneticin was added to eliminate contaminating cells, e.g. conjunctival epithelial cells and fibroblasts. Early passages of conjunctival melanocytes were seeded into multiwell plates and compared with uveal and epidermal melanocytes. Number of dendrites was counted in 500 cells each well. After confluence, cells were detached by trypsin, melanin was extracted and the amount of melanin was measured by spectrophotometer at 475 nm using methods previously described (Invest Ophthalmol Vis Sci 1995; 36: 931).
Conjunctival melanocytes grew well in HU16 medium and could be passaged for 6–10 passages with more than 10 divisions. Several cell lines of human conjunctival melanocytes have been established. Morphologically, conjunctival melanocytes showed many dendrites (average 6.19 dendrites/cell), which was significantly more than that of uveal melanocytes (2.86) and similar to epidermal melanocytes (6.32). Conjunctival melanocytes produced melanin and maintained a constant amount of melanin in vitro (average 0.290 pg/cell), which was significantly greater than that of the uveal melanocytes (0.142 pg/cell) and similar to epidermal melanocytes (0.207 pg/cell).
These in vitro morphological and melanogenesis studies indicate that conjunctival melanocytes are more similar to epidermal melanocytes rather than to uveal melanocytes.
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