A new, simple, nonradioactive, nontoxic in vitro assay to monitor corneal endothelial cell viability

Abstract

Purpose: This study was designed to determine whether Alamar blue could be used to evaluate corneal endothelial cell viability in vitro.

Methods: Alamar blue incorporates a proprietary redox indicator that changes color in response to metabolic activity. Primary rabbit endothelial 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 hours. Sodium azide-killed cells were used as a control. Alamar blue conversion was also compared with [3H]-thymidine incorporation in the presence or the absence of mitomycin C.

Results: Alamar blue reduction demonstrated endothelial cell viability at all cell concentrations compared with that in killed-cell controls. The reduction varied proportionately with cell number and time, showing clearly significant differences. Conversely, [3H]-thymidine uptake demonstrated minimal DNA synthesis and little or no ability to distinguish cell number or viahility.

Conclusions: Alamar blue reduction measures endothelial cell viability and can readily differentiate cell concentrations. It demonstrates several advantages over [3H]-thymidine: It can assay nonproliferating endothelial cell metabolism, it allows rapid assessment of large numbers 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.