Corneal epithelial cells (Japan Tissue Engineering Co., Ltd.)

A 1-mm² sample of the patient's own limbal tissue is taken. The cells are minced and treated with trypsin (0.05% trypsin and 0.91% edetic acid) at 37°C for 3 h. Cells were plated on lethally irradiated 3T3-J2 cells (2.4×10⁴/cm²) and cultured in carbon dioxide (5%) in: Dulbecco's-Vogt Eagle's and Ham's F12 media (3:1 mixture) containing fetal bovine serum (10%), insulin (5 mg/mL), transferrin (5 mg/mL), adenine (0.18 mmol/L), hydrocortisone (0.4 mg/mL), cholera toxin (0.1 nmol/L), triiodothyronine (2 nmol/L), epidermal growth factor (10 ng/mL), glutamine (4 mmol/L), penicillin-streptomycin (50 IU/mL). Subconfluent primary cultures were passaged at a density of 1.5×10⁴ cells/cm² on a circular fibrin substrate in the presence of the feeder layer. At the final stage, corneal epithelial cells are released from the plastic dish with the neutral protease Dispase II (2.5 mg/mL, 45–60 min at 37°C).