Corneas were obtained from the Rocky Mountain Lions’ Eye Bank at 3 to 5 days after harvesting. The ages of the donors ranged from 28 to 58 years. The limbal tissues, including the subconjunctival Tenon’s capsule, were cut into small pieces approximately 2 mm in diameter from clear cornea, which were incubated overnight at 37°C in basal culture medium (DMEM-F12, 1:1; Sigma-Aldrich, St. Louis, MO) with 20 ng/mL of epidermal growth factor (EGF; Wako Pure Chemical Industries, Ltd. Osaka, Japan), 0.02% type IA collagenase (Sigma-Aldrich), B-27
(Table 1) 13 (Invitrogen, Grand Island, NY), 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B. Cells were collected from the incubated tissues in tubes (Sumilon Stem Full; Sumitomo Bakelite Co., Ltd., Tokyo, Japan), allowed to stand in 0.05% trypsin/EDTA (trypsin-EDTA) for 10 minutes at 37°C, and then dissociated into single cells by pipetting. After addition of trypsin inhibitor (Invitrogen), the cells were resuspended in medium with 20 ng/mL of EGF, B-27, 100 U/mL penicillin, 100 μg/mL of streptomycin, and 250 ng/mL amphotericin B. Next, epithelial cells (1 × 10
5 cells/well) were seeded onto pieces of denuded amniotic membrane spread over the culture inserts of six-well culture dishes. The cells were covered with medium for 3 weeks in the culture insert at 37°C in an atmosphere of 5% CO
2/95% air, and the medium was changed daily. In the conventional culture system
10 16 using mouse 3T3-fibroblasts and FBS for HCE equivalent, confluent 3T3 fibroblasts were incubated with 4 μg/mL of mitomycin C for 2 hours at 37°C under 5% CO
2, after which the cells were trypsinized and plated onto plastic dishes of outer wells at a density of 1 × 10
5 cells per well. Corneal limbal epithelial cells (1 × 10
5 cells/well) were seeded onto denuded amniotic membrane spread on the culture inserts, and coculture was performed with 3T3 fibroblasts. The culture medium was DMEM plus Ham’s F12 medium (Sigma-Aldrich) at a 1:1 ratio supplemented with 10% FBS, 0.5% dimethyl sulfoxide, 2 ng/mL EGF, 1 μg/mL recombinant human insulin (Wako Pure Chemical Industries, Ltd.), and 0.1 μg/mL cholera toxin (Sigma-Aldrich). The cell sheets were observed by phase-contrast microscopy.