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J.N. Melvan, R.L. Shearer, C.N. Svendsen, D.M. Gamm; Characterization of Human Prenatal Retinal Pigment Epithelial Cells Cultured Using a Simplified, Serum–Free Method . Invest. Ophthalmol. Vis. Sci. 2006;47(13):894.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a simple, serum–free method for culturing human fetal RPE (hfRPE) to facilitate the investigation, isolation and identification of secreted factors in vitro.
Human prenatal donor eyes (age 54–110 days) were dissected under a microscope and the anterior segment, vitreous and retina were carefully removed. Using jewelers forceps, sheets of RPE with attached choroid were peeled from the underlying sclera and treated with 2% dispase to facilitate separation of the RPE layer. RPE sheets or RPE:choroid explants were chopped into 200 µm sections using a McIlwain® tissue chopper and plated on laminin–coated flasks in defined, serum–free medium containing the supplements N2 or B27. Parallel cultures grown in the absence of serum supplements or the presence of 10% fetal calf serum or 20 ng/ml FGF–2 were also investigated. Established cultures were passaged by Accutase® digestion at regular intervals, at which time total cell counts were obtained for each condition (in triplicate) to determine growth rates. Some cell lines were also maintained in long–term culture to assess viability and monitor their phenotypic characteristics. At specific time points and passage numbers, cultured RPE was examined by immunocytochemistry, Western blot, ELISA and PCR analysis to compare protein and RNA expression and factor secretion.
In the presence of B27 without serum or exogenous mitogens, primary hfRPE could be expanded up to 104–fold over 6 passages (>109 cells generated per eye) and maintained in culture for over one year. Addition of fetal calf serum or FGF–2 increased the overall growth rate of primary hfRPE cultures, but was not required for efficient cell propagation. Differences in cell morphology and pigmentation were observed as a function of passage number, time spent in culture, and media supplementation. Characteristic RPE markers were detected in these hfRPE cultures along with varying levels of secreted factors such as PEDF, VEGF and FGF–2.
Human fRPE can be expanded in defined medium without the addition of serum or mitogens. The optimized culture method described in this study is specifically designed to permit analysis of factor secretion from primary hfRPE in vitro.
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