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Shozo Sonoda, Makoto Shirasawa, Hiroki Otsuka, Toshio Hisatomi, Noboru Arimura, Yasushi Sonoda, Taiji Sakamoto; Assay For Establishment Of Highly Polarized Porcine Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):933.
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Our group reported reproducible method for culturing highly polarized fetal human RPE and show the usefulness of polarized RPE cells (Sonoda et al. Aging 2009, Zhao et al, IOVS 2010). Meanwhile, in many countries, the usage of fetal tissue is illegal. In this study, we attempted to establish the polarized culture system using porcine RPE cells.
Highly polarized porcine RPE cells were grown on TranswellTM filters (12 mm, i.d). Primary cultures of RPE cells on T75 flask were trypsinized, approximately 5×104 RPE cells were seeded on fibronectin-coated TranswellTM. RPE cells were cultured in 10% fetal porcine serum (FPS) containing media for 1 day and changed to 1% FPS thereafter for 4 weeks. The integrity of the RPE monolayer was evaluated by staining for tight junction (TJ) proteins and characterization of well-differentiated RPE cells were evaluated by electron microscopy. Secretion of VEGF-A to the apical and basolateral domains were determined by ELISA after 24 h.
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. The mean concentration of VEGF-A in the apical and basolateral supernatants was 412.5 ± 89.2 pg/ml and 1782.4 ± 221.2 pg/ml, respectively.
Our cell culture procedure produces confluent primary porcine RPE cultures with morphological and physiological characteristics of the native tissue. This system would be useful to better understand the patho-physiology of RPE cells in detail.
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