Long-term culture and growth kinetics of murine corneal epithelial cells expanded from single corneas

Xiaoli Ma, Shigeto Shimmura, Hideyuki Miyashita, Satoru Yoshida, Miyuki Kubota, Tetsuya Kawakita, Kazuo Tsubota

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2716-2721
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume50
Issue number6
DOIs
Publication statusPublished - 2009

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Cornea
Cholera Toxin
Epithelial Cells
Growth
Serum
Air
Keratin-19
Epidermal Growth Factor
Transgenic Mice
Population
Cell Differentiation
Cultured Cells
Epithelium
Western Blotting
Immunohistochemistry
Polymerase Chain Reaction
Messenger RNA
involucrin

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Long-term culture and growth kinetics of murine corneal epithelial cells expanded from single corneas. / Ma, Xiaoli; Shimmura, Shigeto; Miyashita, Hideyuki; Yoshida, Satoru; Kubota, Miyuki; Kawakita, Tetsuya; Tsubota, Kazuo.

In: Investigative Ophthalmology and Visual Science, Vol. 50, No. 6, 2009, p. 2716-2721.

Research output: Contribution to journalArticle

Ma, Xiaoli ; Shimmura, Shigeto ; Miyashita, Hideyuki ; Yoshida, Satoru ; Kubota, Miyuki ; Kawakita, Tetsuya ; Tsubota, Kazuo. / Long-term culture and growth kinetics of murine corneal epithelial cells expanded from single corneas. In: Investigative Ophthalmology and Visual Science. 2009 ; Vol. 50, No. 6. pp. 2716-2721.
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AU - Ma, Xiaoli

AU - Shimmura, Shigeto

AU - Miyashita, Hideyuki

AU - Yoshida, Satoru

AU - Kubota, Miyuki

AU - Kawakita, Tetsuya

AU - Tsubota, Kazuo

PY - 2009

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N2 - 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.

AB - 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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