New non‐toxic fluorescent marker for quantifying the viability of corneal endothelial cells

Abstract

Aims/Purpose: Viability markers are essential for cellular research and tissue transplantation. However, existing options such as Trypan Blue lack specificity, and some clinically used markers are cytotoxic. Therefore, we synthesized the compound F4267, a non‐fluorescent marker that releases fluorescein upon hydrolysis. The aim of this study was to evaluate the quality of F4267 labelling of endothelial cells and its cytotoxicity in immortalized cells and organ‐cultured endothelium.Methods: We compared the marking capability of F4267 and Calcein‐AM using immortalized cells and endothelium from corneal pairs. The quality of fluorescent labelling was assessed using F4267 at 4 μM and Calcein‐AM at 4 μM. Cytotoxicity of F4267 was evaluated at varying concentrations of 40 and 1000 μM on cells, and at 40 μM on corneal endothelium, with single, repeated, or prolonged exposures. The final density and viability of corneal endothelium were measured using HEC1 staining.Results: F4267 (40 μM) exhibited fluorescent labelling quality equivalent to Calcein‐AM (4 μM), allowing clear differentiation between living, dying, and acellular areas. In HCE‐2 cells, cytotoxicity was reduced by 19% with F4267 (40 μM) compared to Calcein‐AM (4 μM). Prolonged exposure (24 h) to HCEC‐B4G12 cells showed no cytotoxicity with F4267 (40 and 100 μM), but resulted in destruction of the cell layer with Calcein‐AM (4 μM). F4267 (40 μM) also demonstrated significantly lower cytotoxicity, reducing mortality by 10% (single exposure) and 17% (repeated exposure) in organ‐cultured corneas.Conclusions: F4267 shows promise as a marker for assessing viable endothelial cells in organ culture, providing a safe alternative to existing markers. Its release of fluorescein with excellent cell tolerance supports its clinical potential. The integration of viable endothelial cell density assessment would enhance the value of corneal banks. Further validation is required, as well as exploration of broader applications.Reference1. Pipparelli et al., IOVS 2011.