Human corneal epithelial cells (HCECs) were isolated and cultured from postmortem donor corneas unsuitable for transplantation (Banque Nationale d’Yeux du CHUQ), as described previously.
37 Briefly, corneas were dissected from the ocular globes with curved scissors (Storz, St. Louis, MO), and the limbus was separated from the central cornea with a 7.5-mm diameter trephine (Pilling Weck, Markham, ON, Canada). The limbal ring was incubated in 2 mg/mL dispase II (Roche Diagnostics, Laval, QC, Canada) in HEPES buffer (MD Biomedicals, Montreal, QC, Canada) for 18 hours at 4°C. The epithelium was mechanically removed from the stroma with forceps under a dissecting microscope (model SMZ-2T; Nikon, Montreal, QC, Canada), cut into small pieces with a scalpel, and centrifuged for 10 minutes (200
g) at room temperature. The HCEC pellet was then seeded in tissue culture flasks along with irradiated (60 Gy) murine Swiss-3T3 fibroblasts (ATCC, Rockville, MD). Primary cultures (P0) were subcultured up to the fourth passage (P4). Human corneal fibroblasts were isolated from the stromal portion of the cornea left after dispase digestion and removal of the epithelium and endothelium. Briefly, the stroma was incubated for 3 hours at 37°C in a collagenase H solution (0.125 U/mL; Roche Diagnostics). After a 10-minute centrifugation (room temperature), cells were seeded in 25-cm
2 tissue culture flasks and cultured, as previously described.
37 Fibroblasts were used in all experiments between the fifth and seventh passages. Human skin fibroblasts were obtained from the dermal portion of adult breast skin and were cultured, as described previously.
40 41 Each cell type was grown under 8% CO
2 at 37°C, and the culture medium was changed three times a week.