Endothelial cells were cultured according to previously published methods.9- 12 Corneal endothelial cells from fresh banked human donor tissue not suitable for transplantation (Central Florida Lions Eye and Tissue Bank, Tampa, Florida, and Tissue Banks International, Baltimore, Maryland) were harvested attached to the Descemet membrane on or before the seventh day after death. The age of the donor tissue was 56 years. After overnight incubation of the Descemet membrane/endothelial cell complex in OptiMem-I (Gibco, Invitrogen Corp, Carlsbad, California) containing 8% fetal bovine serum, the complex was centrifuged and washed in Hank balanced salt solution (Mediatech, Inc, Herndon, Virginia). Next, the endothelial cells and Descemet membrane complex were incubated for 1 hour in 0.02% of EDTA solution, stirred vigorously with a flame-polished pipette to disrupt cell junctions, centrifuged for 5 minutes at 3000g (g-force), and seeded onto culture plates coated with FNC coating mix (Athena Enzyme Systems, Baltimore) containing bovine fibronectin (10 mg/mL) and bovine type I collagen (35 mg/mL). The cells were cultured in OptiMem-I media supplemented with 8% fetal bovine serum, calcium chloride (200 mg/L), chondroitin sulfate (0.08%), ascorbic acid (20 μg/mL), pituitary extract (100 μg/mL), epidermal growth factor (5 ng/mL), nerve growth factor (20 ng/mL), gentamicin (1:200), penicillin (1:100), streptomycin (1:100), and amphotericin (1:100) under 10% carbon dioxide. Medium was changed every 2 days. At confluence, the cells were split 1 to 3, and passage 4 cells were used for experiments. The experiments were performed at 70% confluence.