title: Long-term culture and growth kinetics of murine corneal epithelial cells expanded from single corneas.
abstract: OBJECTIVE
  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-Ca(2+) 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. Colony-forming 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 Ca(2+)-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 Ca(2+) 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.
authors.affiliation: Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Ma
authors.forename: Shigeto
authors.initials: S
authors.lastname: Shimmura
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Miyashita
authors.forename: Satoru
authors.initials: S
authors.lastname: Yoshida
authors.forename: Miyuki
authors.initials: M
authors.lastname: Kubota
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kawakita
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tsubota
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002452:Cell Count; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D015925:Cryopreservation; D016895:Culture Media, Serum-Free; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D007150:Immunohistochemistry; D053530:Keratins, Type I / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
chemical_list: D016895:Culture Media, Serum-Free; D053530:Keratins, Type I; D010750:Phosphoproteins; D011498:Protein Precursors; D012333:RNA, Messenger; D015534:Trans-Activators; C486419:Trp63 protein, mouse; C032253:involucrin