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M. Bonne, V. Dedes, P. Labalette, L. Devisme, B. Gosselin, J. Rouland, J. Kerr Conte; Comparative Study of Cultured Limbal Epithelial Cells on a 3T3 Feeder Layer or in Defined Medium . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1350.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Autologous cultured epithelial cells can restore damaged corneas. The techniques implied, however, are still being developed. The aim of this study was to compare different types of culture for limbal epithelial cells. Methods: Epithelial cells were obtained from trypsinized limbal rings normally discarded after keratoplasty (n=30). Epithelial cells were cultured in plastic dishes on a 3T3 feeder layer or in serum free medium (K-SFM GIBCO® ). We measured Colony Forming Efficiency and kinetics parameters of cultured epithelial cells, and defined their phenotype by immunostaining with monoclonal antibodies to keratin and mucins. We then made histologic sections of cultured epithelium. Results: For 100 epithelial cells cultured, the number of colonies greater than 50 cells was 0,44 % ± 0,57% (n=6) after 12 days of culture with 3T3 and 8,22 % ± 3,96% (n=5) in serum free medium. Colonies appeared earlier in serum free medium (3 ± 1 days) than in cultures with 3T3 (7 ± 2 days). We did not observe any difference in the expression of Muc1 or cK19 before and after culture, but noted that cK3 and Muc5AC increased. The histologic sections showed a pluristratified epithelial dysmaturation in cultures with 3T3 and a regular unistratified epithelium in serum-free medium culture. Conclusions: Large colonies of cultured epithelial cells expressing corneal and limbal phenotype can be easily and quickly obtained with a serum-free medium usually used for keratinocyte culture.
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