Purpose: To develop a reproducible procedure for the long-term culture of corneal epithelial cells from a single mouse cornea. Methods: Corneal limbal explants of C57BL6/J mice were cultured in serum-free, low-Ca2+ medium supplemented with EGF and cholera toxin. Epithelial cells were subcultured at a 1:3 split until passage (P)4 and at lower densities after P4. Colonyforming efficiency, population-doubling times, and population doublings were determined. The expression of p63, keratin (K)19, K12, and involucrin was analyzed by RT-PCR, immunocytochemistry, and Western blotting. Differentiation potential was examined by switching the medium to serum or high Ca2+ -containing medium. Stratification ability was analyzed by air-lift culture. Results: Thirty of 32 (93.8%) corneal explants were successfully subcultured to P1. Cultures without cholera toxin did not proliferate past P2 (n= 12), but 55% of cultures supplemented with cholera toxin achieved P4 (n = 20). After P4, cells were stably subcultured over 25 passages. Colony-forming efficiency increased from 9.7%±2.6% at P5 to 29.0%±3.3% at P20. The cells showed cobblestone appearance and expressed p63, K19, and involucrin but were negative for K12. Serum and high Ca2+ induced differentiation, and cells cultured in DMEM/F12 with serum showed K12 mRNA expression. Stratified epithelium was formed by air-lifting. Conclusions: With this procedure, corneal epithelial cells from a single cornea can be cultured long term and can retain the potential to differentiate and stratify. This procedure can be a powerful tool for studies that require comparison of corneal epithelial cells from normal and transgenic mice in vitro.
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