Measuring the viability of crystalline lens epithelial cells by triple Hoechst-Ethidium-Calcein-AM staining

To date, the assessment of lens epithelial cell viability has been proposed only in cell cultures or isolated capsule models. This study aimed to develop a method for quantifying the viability of epithelial cells on whole ex vivo crystalline lenses by triple labeling Hoechst 33342, ethidium homodimer, and calcein-acetoxymethyl (HEC).

Two models of induced cell death were used to study the performance and potential applications of the technique. First, ten fresh pairs of six-month-old porcine lenses were retrieved. On one lens of each pair, an easily identifiable localized lesion was induced by a calibrated cryo-application, while the other remained intact. Both lenses of each pair were incubated for 1 h at 20 °C in an HEC mixture. Ten other pairs of lenses were used in the second experiment. On one lens of each pair, a diffuse epithelial lesion was induced by incubation in staurosporine (STS) solution (0.5 µM in CorneaMax) for 24 h at 37 °C. The other lens of each pair was incubated in CorneaMax solution without STS for 24 h at 37 °C. The day after, both lenses of each pair were incubated for 1 h at 20 °C in an HEC mixture. Images were acquired with a macroscope (macro zoom) and analyzed with ImageJ. Calcein-AM and ethidium images were used to calculate the area covered by living epithelial cells. Hoescht images allowed us to count cell nuclei per unit area. Viable epithelial cell density (vECD) was defined as the number of viable cells per unit area. Different strategies were developed to reduce background noise.

There was no interfering lens autofluorescence for the exposure times used. The vECD median was 2,840 cells/mm² [10th-90th percentiles = 2,479-3,494] for cryo-injured lenses versus 3,364 cells/mm² [2,919-3,739] for healthy lenses (p = 0.002). The vECD median was 3,804 cells/mm² [10th-90th percentiles = 2,922-4,862] for lenses treated with STS versus 3,896 cells/mm² [3,169-4,980] for healthy lenses (p = 0.002).

Thanks to simple sample preparation, triple HEC staining allows fluorescence imaging of a large series of a whole lens to respect the architecture of the epithelium. It will be particularly useful for cytotoxicity studies of new therapies targeting the lens.