Cadaver donor corneal-scleral rims were obtained with appropriate research consent from Moorfields Lions Eye Bank. Ethical permission for this study was obtained from the Research Ethics Committee (UK, ref. no. 10/H0106/57-11ETR10), and all tissue was handled in accordance with the Declaration of Helsinki. Corneas were stored by organ culture method at ambient temperature after enucleation for 4 to 6 weeks before hLEC isolation.
Human limbal epithelial cells were isolated in either corneal epithelial cell medium (CM) containing Dulbecco's modified Eagle's medium-F12 (DMEM:F12) basal medium (3:1 dilution), 10% fetal bovine serum, 1% antibiotic–antimycotic, epidermal growth factor (EGF; 10 ng/mL; Life Technologies Ltd., Paisley, UK), hydrocortisone (0.4 μg/mL), insulin (5 μg/mL), adenine (0.18 mM), transferrin (5 μg/mL), T3 (2 nM), cholera toxin (0.1 nM; Sigma-Aldrich, Dorset, UK), or in stromal stem cell medium (SM) containing DMEM-MCDB-201 (3:2 dilution; Sigma-Aldrich), 2% fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 μg/mL), gentamycin (50 μg/mL), insulin (10 mg/mL), transferrin (5.5 mg/mL), selenous acid (6.7 ng/mL; 1× ITS; Life Technologies), albuMAX-I (1 mg/mL; Life Technologies Ltd.), dexamethasone (10 nM), EGF (10 ng/mL), l-ascorbic acid 2-phosphate (120 μM), cholera toxin (100 ng/mL), platelet-derived growth factor (10 ng/mL; Sigma-Aldrich). Isolation of hLECs was carried out with or without a feeder (F) layer of 3T3 cells (3T3-Swiss albino cells, catalog no. CCL-92; American Type Culture Collection, Manassas, VA, USA) whose growth was arrested with 4 μg/mL mitomycin C (Sigma-Aldrich) for 2 hours at 37°C.