Cell viability assay in corneal endothelium

Abstract

[Abstract] Introduction: Endothelium is the inner layer of the cornea, which must be viable for transplanting. The limited availability of corneas makes necessary the developing of preservation techniques that allow a long storage without losing endothelial viability.Objectives: Optimization of a cell viability assay in preserved corneas.Methods: One half of an endothelium from a cornea that was storage in hypothermic conditions and an endothelium of a cryopreserved cornea were stained with LIVE/DEAD imaging kit and Hoechst. The other half of endothelium was the negative control. Corneal endothelia were imaged using a fluorescence microscope.Results: Four sort of cells were visualized on both endothelia: viable cells with high esterase activity, intermediate cells with low esterase activity, non-viable cells without esterase activity, and cells only stained by Hoechst. Conclusions: Triple stain is effectiveto detect different sort of cells in endothelium of preserved corneas, included viable cells, depending on their esterase enzymatic activity and on cell and nuclear membrane damage.