Assay for establishment of highly polarized retinal pigment epithelial cells

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Our group attempted to establish a reproducible method for culturing highly polarized RPE and tried to elucidate the pathophysiological mechanism of retinal disease using polarized culture RPE.Highly polarized RPE cells were grown on Transwell^<TM> filters(12 mm, i. d). Primary cultures of RPE cells on T75 flask were seeded on fibronectin-coated TranswellTM. RPE cells were cultured in 1% FBS for more than 4 weeks. As in native tissue, porcine RPE cells formed a monolayer, were well pigmented, and were arranged in a regular hexagonal array. The integrity of RPE layer was confirmed by expression of TJ proteins ZO-1 and occluding and well developed microvilli, localization of pigment on the apical side, nuclei on basal side, and presence of tight-junctional complexes by electron microscopy. Well-differentiated RPE cells secreted VEGF-A preferentially to the basolateral side of the tissue. With respect to application of polarized culture RPE system, after stimulation with TNF-a, TER decreased about 15% of reduction in comparison to control at 24 hours, which was abolished by anti-TNF-a antibody. This reduction was inhibited by SB203580 inhibitor, but not by any other inhibitors.