A NEW, SIMPLE, NONRADIOACTIVE, NONTOXIC IN-VITRO ASSAY TO MONITOR CORNEAL ENDOTHELIAL-CELL VIABILITY

Purpose. This study was designed to determine whether Alamar blue coul d be used to evaluate corneal endothelial cell viability in vitro. Met hods. Alamar blue incorporates a proprietary redox indicator that chan ges color in response to metabolic activity. Primary rabbit endothelia l cells were subcultured on 96-well plates at densities ranging from 1 ,250 to 40,000 cells per well. After 12 hours' incubation, Alamar blue was added to each well and absorbance measured hourly from 1 to 9 hou rs. Sodium azide-killed cells were used as a control. Alamar blue conv ersion was also compared with [H-3]-thymidine incorporation in the pre sence or the absence of mitomycin C. Results. Alamar blue reduction de monstrated endothelial cell viability at all cell concentrations compa red with that in killed-cell controls. The reduction varied proportion ately with cell number and time, showing clearly significant differenc es. Conversely, [H-3]-thymidine uptake demonstrated minimal DNA synthe sis and little or no ability to distinguish cell number or viability. Conclusions. Alamar blue reduction measures endothelial cell viability and can readily differentiate cell concentrations. It demonstrates se veral advantages over [H-3]-thymidine: It can assay nonproliferating e ndothelial cell metabolism, it allows rapid assessment of large number s of samples, it can differentiate endothelial cell concentrations, it is nontoxic, it is nonradioactive and allows for simple disposal, it is less costly, and it allows for continuous monitoring of endothelial cell metabolism and viability.