This research followed the tenets of the Declaration of Helsinki involving human subjects and was approved by the institutional review committee of Taipei Veterans General Hospital. The HCECs were isolated and cultured as described previously.19 All of the cell culture supplements were purchased from Sigma-Aldrich Corp (St Louis, Mo) unless otherwise noted. The 25 human corneas used in this study were obtained from National Disease Research Interchange, Philadelphia, Pa, and were from donors aged 55 to 80 years, and the corneas had been stored in Optisol-GS solution (Bausch and Lomb Surgical, Irvine, Calif) at 4°C. For the isolation of endothelial cells, the Descemet membrane–corneal endothelium complex was aseptically stripped from the stroma and digested using 1.2 U/mL of a grade II neutral protease (Dispase II; Roche Diagnostics, Indianapolis, Ind) in Hanks balanced salt solution (Gibco, Grand Island, NY) for 1 hour at 37°C. The HCECs were pelleted and resuspended in regular growth medium containing modified Eagle medium (OptiMEM; Gibco) as a basal medium, 15% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), 40 ng/mL of bovine pituitary fibroblast growth factor, 5 ng/mL of human recombinant epidermal growth factor (Upstate Biotechnology, Lake Placid, NY), 20 ng/mL of nerve growth factor (Biomedical Technologies, Stoughton, Mass), 20 μg/mL of ascorbic acid, 0.005% human lipids, 0.2 mg/mL of calcium chloride, 0.08% chondroitin sulfate, 1% RPMI 1640 vitamins solution, 50 μg/mL of gentamicin (Gibco), and 1% antibiotic-antimycotic solution (Biological Industries). The cultures were incubated in a humidified atmosphere of 5% carbon dioxide at 37°C. The medium was changed every other day. After 1 week in culture, confluent cell monolayers were subcultured by treating with trypsin-EDTA (Gibco) for 2 minutes and seeded at a 1:3 split ratio. Only second-passage HCECs were used during all experiments.