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L. Zheng, C. M. Blaha, S. E. Beck, D. Myung, J. R. Cochran, C. Ta; Morphology and Phenotype Comparison of Primary Rabbit Epithelial Cell Cultures in Serum-Fortified and Serum-Free Media. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3368.
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© ARVO (1962-2015); The Authors (2016-present)
This study characterized the growth of rabbit corneal epithelial cells (CECs) in various media commonly used in corneal epithelial cell culture.
CECs were isolated from enucleated New Zealand white rabbit eyes by incubation in dispase solution. CECs in primary culture were grown to confluence in Keratinocyte Serum-Free Medium (KSFM), Supplemental Hormonal Epithelial Medium (SHEM), Medium 199 (M199), and DMEM/F12. Cells that were expanded in KSFM were then cultured in either SHEM or KSFM, with or without serum and EGF. Immunofluorescence stainings of cytokeratin 3/12 and claudin-1 were used to assess epithelial phenotype and presence of tight junctions.
Primary culture CECs reached confluence in all four media, but varied in morphology. Cells grown in KSFM retained the most epithelial-like phenotype. CECs appeared fibroblastic in SHEM, and large and flat in both DMEM/F12 and M199. Cells that were expanded in KSFM proliferated more slowly in the absence of EGF, and did not proliferate at all when neither EGF nor serum was present. In SHEM, CECs displayed a more epithelial-like morphology in the presence of EGF and did not proliferate when both EGF and serum were absent. All KSFM and SHEM variants stained positive for cytokeratin 3/12. Cells grown in KSFM (<0.1 mM calcium) were able to express claudin-1.
The optimal media for isolating and expanding primary rabbit corneal epithelial cells was KSFM. This media promoted the most epithelial-like phenotype and was able to support tight junction formation despite a low calcium concentration. Presence of EGF and serum in media such as KSFM and SHEM clearly enhanced cell growth.
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