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A. Ahmado, A. A. Vugler, J. Lawrence, P. Coffey; Growth Media Related Pigmentation of the Cultured Human Retinal Pigment Epithelium Cell Line ARPE19. Invest. Ophthalmol. Vis. Sci. 2008;49(13):489.
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The retinal pigment epithelium undergoes de-differentiation and quickly loses its pigmentation in culture, together with other markers of differentiation (e.g. RPE65, CRALBP). Na+/K+ ATPase is a marker that is still present in cultured RPE but its distribution is affected whereby it becomes basolateral rather than apical.
Various passages (p22 - p28) of the human retinal pigment epithelial cell line ARPE19 were cultured on a variety of substrates and using several variations of growth media and fetal bovine serum concentrations. Cultures were fed twice per week. Samples were fixed and stained for immunohistochemistry and electron microscopy. Immunostaining was analysed using a confocal microscope.
Macroscopic pigmentation was observed in cultured ARPE19 when DMEM x1 High Glucose with 1% FBS concentration was used as growth medium. Pigmentation was never observed with DMEM:F12 with 10% FBS concentration. Pigmentation in culture developed on glass, polystyrene plates, cellulose and polyester filters, with or without a coating of extacellular matrix substitute. In all cases, pigmentation was heterogenous and distributed in a mosaic. Pigmentation required at least 4-6 weeks in culture. Pigmented cells expressed apical Na+/K+ ATPase compared to their neighbouring non-pigmented cells which had basolateral expression. Pmel 17 positive staining was abundant in pigmented cultures. Electron microscopy confirmed the prescence of immature melanosomes which were varied in density and distribution.
A growth medium has been identified which induces in-vitro re-pigmentation in the human retinal pigment epithelial cell line ARPE19. Further studies are necessary to characterise the pathways triggering re-pigmentation and whether melanosomes are capable of full maturation and correct distribution in this in-vitro scenario.
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