title: New technique for culturing corneal epithelial cells of normal mice.
abstract: OBJECTIVE
  To describe a new method of culturing mouse corneal epithelial cells (MCECs).
  
  
  METHODS
  MCECs were isolated from C57/BL6 mouse corneas and cultured on type-I collagen-coated plastic dishes in low-calcium progenitor cell targeting medium (CnT-50). Expression of the mRNAs of N-terminal truncated isoform of p63 (DNp63), cytokeratin 12 (K12), and cytokeratin 14 (K14) were determined by reverse transcription-polymerase chain reaction (RT-PCR). To examine the differentiation capabilities, passage 3 (P3) MCECs at confluence were subcultured on amniotic membrane (AM) in a differentiation medium (CnT-30) until confluence. At confluence, 1 mM calcium was added and cultured for 4 more days. The expression of K12 in the stratified MCECs was analyzed by immunostaining.
  
  
  RESULTS
  The MCECs cultured in CnT-50 proliferated until at least P10. The number of cells at confluency at P3 was 61.8 (SD +/-9.4, n=5) times that at P0. MCECs cultured on AM in CnT-30 with addition of calcium were stratified up to two to three layers, and the stratified MCECs expressed K12. DNp63 mRNA was continuously expressed throughout the different passages, and K12 mRNA was detected in P0 cells and the stratified MCECs on AM.
  
  
  CONCLUSIONS
  Cultured MCECs maintain their proliferation and differentiation capabilities as well as their corneal epithelial cell characteristics. These results suggest that MCECs produced by this culturing method provide a useful experimental model which can enable further development of research of the corneal epithelium.
authors.affiliation: Department of Ophthalmology and Regenerative Medicine, Ehime University School of Medicine, Shitsukawa, Toon, Ehime, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Ryuji
authors.initials: R
authors.lastname: Yoshioka
authors.forename: Atsushi
authors.initials: A
authors.lastname: Shiraishi
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Ohashi
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology*; D007150:Immunohistochemistry; D007633:Keratins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008858:Microscopy, Phase-Contrast; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
chemical_list: D010750:Phosphoproteins; D015534:Trans-Activators; C486419:Trp63 protein, mouse; D007633:Keratins
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