Primary CEC Culture
CECs retrieved from control tissue and from subjects diagnosed with FECD were cultured in accordance with a dual media approach described by Peh et al.25 In brief, donated tissue comprising DM with attached endothelial cells was incubated in 0.2% collagenase type I powder in M5 media (Life Technologies) for 3 hr at 37°C to dislodge CECs from the DM. CECs were centrifuged and re-suspended again in M5 media to allow for cell adherence and stabilization. Media M5 contained Human Endothelial-SFM (Life Technologies) supplemented with 5% FBS, 1% antibiotic/antimycotic, and 0.1% selective ROCK inhibitor Y-27632 (AdooQ BioScience). Cells were seeded in cell culture ware pre-coated with FNC coating mixture (United States Biological). After 24 hr, to promote proliferation, culture media was replaced with M4 media containing Ham’s F-12 Nutrient Mix GlutaMAX Supplement (Life Technologies)/Medium 199 GlutaMAX Supplement (Life Technologies), 20 μg/mL ascorbic acid, 1% insulin-transferrin-selenium (Life Technologies), 5% FBS, 1% antibiotic/antimycotic, 10 ng/mL bFGF (R&D Systems), and 0.1% selective ROCK inhibitor Y-27632 (AdooQ BioScience). Throughout culture, cells were kept in an incubator at 37°C, 5% CO2 and medium was refreshed every 48 hr until the cells showed appropriate confluence for experimentation or passage. Cells were passaged a maximum of two times prior to any experiment being performed.