Isolation and Culture of Human Corneal and Conjunctival Epithelial Cells

Human primary CoEpiCs and CjEpiCs were obtained from donor corneas (CorneaGen, Seattle Eye Bank, Seattle, WA, USA). The cells were isolated and cultured following a previously described method [16 (link)] with slight modifications. The corneal and conjunctival epithelium were peeled off after overnight treatment with dispase type II (Godo Shusei, Tokyo, Japan) at a concentration of 1000 PU/mL at 4°C. The epithelial cells were then dissociated into single cells by incubation with TrypLE™ Express (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min at 37°C and seeded into a single well of a 6-well plate. The CoEpiCs and CjEpiCs were cultured at 37°C, 95% humidity, and 5% CO₂ in complete medium [17 (link)], consisting of Dulbecco’s modified Eagle’s medium and Ham’s F-12 media (DMEM/F12, 1:1 mixture) (Thermo Fisher Scientific), B-27™ supplement (2%) (Thermo Fisher Scientific), Rho-kinase inhibitor Y-27632 (10 μM) (Selleck Chemicals, Houston, TX, USA), keratinocyte growth factor (10 ng/ml) (Thermo Fisher Scientific), and penicillin-streptomycin (50 IU/ml) (Nacalai Tesque, Kyoto, Japan). The cells were used for subsequent experiments after one passage.