Abstract
Aim/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. We have therefore 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 F4267 and Calcein-AM marking capability were compared using immortalized cells and the endothelium from corneal pairs. The quality of fluorescent labelling was assessed with F4267 at 4µM and Calcein-AM at 4µM.F4267 cytotoxicity was assessed at varying concentrations of 40 µM and 1000 µM on cells and Calcein-AM at 40µM on corneal endothelia with single, repeated or prolonged exposures. The final density and viability of corneal endothelia was measured using HEC1 staining.
Results F4267 (40µM) showed a fluorescent labelling quality equivalent to Calcein-AM(4µM), allowing clear differentiation between living, dying and acellular areas. In HCE-2 cells, cytotoxicity was reduced by 19% between F4267 (40µM) and Calcein-AM (4µM). Prolonged exposure (24h) on HCEC-B4G12 cells showed no cytotoxicity with F4267(40 and 100µM) but destruction of the cell layer with Calcein-AM (4µM). F4267 (40µM) also showed significantly lower cytotoxicity, reducing mortality by 10% (single exposure) and 17% (repeated exposure) on corneas in organculture
Conclusion F4267 is a promising marker for the assessment of viable endothelial cells in organoculture, offering a safe alternative to existing markers. Its fluorescein release with excellent cell tolerance supports its clinical potential. The integration of viable endothelial cell density assessment would improve the value of corneal banks. Further validation is required, as well as opening up to wider applications.
Reference
Pipparelli, et al. IOVS 2011.
