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C. Engler, C. Kelliher, C. L. Speck, A. S. Jun; Assessment of Corneal Endothelial Marker Expression in Transformed Murine Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1816.
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© ARVO (1962-2015); The Authors (2016-present)
To assess corneal endothelial marker expression in transformed murine corneal endothelial cells.
Immortomice with thermo-sensitive SV40 T-antigen were sacrificed after anesthesia and globes were excised. After excision of the corneas, corneal buttons were submerged in culture medium, and small strips of Descemet’s Membrane (DM) were removed with a jewellers forceps. DM strips were incubated at 33° C and 5% CO2. Immunostaining for ZO-1 and JAM-1 was performed for functional assessment of the cell monolayer. After extraction of total RNA of DM and attached endothelial cells real-time reverse transcriptase (RT) - PCR for Col8A2 was performed with harvested DM strips, newly cultured cells and with cells held in culture for several passages.
Cells started growing out of DM after approximately 7-10 days and formed a monolayer of polygonal cells. Immunostaining for ZO-1 and JAM-1 showed intact expression of cell adhesion molecules. Real-time RT-PCR showed expression for Col8A2. Alterations in ZO-1, JAM-1, Col8A2 expression over time in culture will be presented.
Even though technically challenging, it is possible to culture murine corneal endothelial cells using the Immortomice strain. These cells retain the expression of corneal endothelial cell markers which supports their usefulness as a model for research purposes.
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