Isolation and Culture of Primary Mouse Retinal Pigment Epithelial (RPE) Cells with Rho-Kinase and TGFβR-1/ALK5 Inhibitor

: BACKGROUND Primary RPE cells could be a reliable model for representing in vivo status of RPE compared with cell lines. We present a protocol for in vitro isolation and culture of primary RPE cells from C57BL mice. MATERIAL AND METHODS We used C57BL mice ages 7 days to 4 months. The RPE layer was separated from the neural retina layer by digestion with 2% Dispase for 45 min and scraped off from the choroid after 25-min incubation in 37°C. Collected RPE sheets were gently pipetted up into smaller sheets. RPE sheets were transferred into well plates and cultured in vitro for 2 weeks. To inhibit epithelial-mesenchymal transition (EMT) of RPE cells, we used Y27632 and Repsox to treat cultured primary RPE cells. RESULTS RPE cells isolated from C57BL mice maintained pigmented and hexagonal morphology in culture. However, long-term in vitro culture lead to the periphery cells of a RPE sheet becoming mesenchymal-like cells. In contrast to the control group, Y27632 and Repsox, which are inhibitors of Rho-kinase or TGFβR-1/ALK5, promoted primary RPE cells to maintain epithelial-like morphology and eventually become confluent. CONCLUSIONS RPE cells isolated from C57BL mice could be a powerful cell model to study the biological function of RPE. Especially, C57BL mice with different defective genetic background resulting in ocular diseases, would expand the genome type of RPE cells. The method presented here could be an efficient and applicable technique to obtain large numbers of primary RPE cells that maintain some characteristics of in vivo RPE.