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R. L. Shearer, L. S. Wright, G. Sabat, J. N. Melvan, C. N. Svendsen, D. M. Gamm; Human RPE Cultures Grown in Serum-Free Conditions Retain Appropriate Cellular Polarity and Secretory Capacity. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2531.
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To determine the apical-basal polarity and secretory capacity of human fetal RPE (hRPE) monolayer cultures grown and maintained in serum-free, defined medium.
RPE explants from human prenatal donor eyes (54-110 days post-conception) were initially grown as suspended spheres in defined serum-free medium. Monolayer cultures obtained from plated RPE spheres were maintained long-term in serum-free media. Cultured hRPE was examined by immunocytochemistry and confocal microscopy to ascertain cell polarity. To assess secretory capacity, RPE conditioned media was collected over a 24 hr period and concentrated. Proteins were precipitated, reconstituted, and run on an IPG strip with an isoelectric range of pH 3 to 10. The strip was then loaded onto a 8-16% gradient gel and proteins were separated in the second dimension. After staining, spots were picked for "in gel" digestion and run on a MALDI TOF-TOF mass spectrometer. Raw data was deconvoluted using GPS Explorer software and submitted for peptide mapping and MS/MS ion search analysis against a non-redundant NCBI database with an in-house licensed Mascot search engine.
Immunocytochemistry and confocal microscopy confirmed the proper apical-basal orientation of these monolayer hRPE cultures. Specifically, ezrin and Na,K-ATPase were expressed predominantly on the apical surface, bestrophin was localized to the basolateral surface, and CRALBP and RPE65 were present within the cytoplasm. In addition, ZO-1 and occludin were present within intercellular junctions. The detectable secretome of the hRPE cultures consisted of several proteins known to be released by RPE, including factors involved in metal binding, oxidant regulation, protein degradation and signaling.
These results demonstrate that hRPE monolayers cultured in the total absence of serum exhibit appropriate apical-basal orientation and secrete numerous proteins involved in the maintenance of outer retinal health and function.
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