Isolation and Culture of RPE Cells

RPE cells were isolated according to the method reported by Shang et al. [24 (link)], with some modifications. The enucleated eyes were digested for 35 min at 37 °C in dispase II solution (2 % dispase II [Thermo Fisher Scientific], 10 mM HEPES [pH 7.4], 30 mM NaCl in DMEM [4.5 g/L glucose]). Digested eyeballs were washed twice with growth medium (DMEM [4.5 g/L glucose] containing 10 % FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2.5 mM l-glutamine). An incision was made near the ora serrata of the eyes and the anterior segment of the eye was removed. The remaining posterior eye was transferred to a new growth medium, and the neurosensory retina (NSR) was removed. The RPE–choroid complex was transferred to a new growth medium and RPE cell sheets were peeled from the choroid. Isolated RPE cells were washed twice with growth medium and centrifuged (800×g, 4 °C, 5 min).