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X. Ma, S. Shimmura, H. Miyashita, T. Kawakita, K. Tsubota; Long-Term Culture of Murine Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3369. doi: https://doi.org/.
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To develop a reproducible technique for the long-term culture of corneal epithelial cells from a single mouse.
The initial stage of culture was done using explants of corneal limbal tissue of C57BL6/J mice cultured in serum-free low calcium medium (defined KSFM, Invitrogen). After cells reached subconfluence, the epithelial sheets were subcultured at 1:3 split until passage 4 (P4) cultures. From P4, cells were subsequently serially passaged at a low cell density (700 cells per cm2). Cells from each animal were kept separate throughout the culture procedure. Colony-forming efficiency (CFE) and cumulative number of cell population doublings (PDs) were determined. The expression of corneal epithelial cell markers p63, keratin 19 (K19), keratin 12 (K12) and involucrin was investigated by RT-PCR analysis and immunocytochemistry. The ability to form stratified epithelial sheets was analyzed by exposing cells to the air-medium interface.
Nineteen of 20 cornea explants (95%) were successfully subcultured to P1, and the success rate decreased to 55% by P4. However, after P4, cells were stably subcultured with the oldest line reaching P20. CFE at P5 was 9.67± 0.41% (mean ±SD, n=3) and PDs at P15 was 48.94±3.57 (mean ±SD, n=3). The cells showed typical cobblestone appearance, expressed p63, K19 and involucrin, but were negative for K12. The cells became stratified when cultured in an air-medium interface.
Using this technique, corneal epithelial cells from a single mouse can be cultured long-term and form stratified epithelia. This technique can be a powerful tool for studies that require comparison of corneal epithelial cells from normal, transgenic, or knockout mice in vitro.
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