Evaluation of pan‐epithelial viability in the whole porcine lens

Purpose: The lens has a monolayer of epithelial cells on its anterior surface that contributes to its homeostasis. A quantification of the viability of these cells is essential to evaluate the safety of new therapies targeting the lens (cataract, presbyopia). The observation of this whole tissue is made difficult by its optical properties (transparency and refraction) and only simplified models have been used until now to measure epithelial viability (primary cell cultures or partial surgical specimens). Aims: To develop a method to quantify the viability of epithelial cells on whole ex vivo lenses. Methods: Ten fresh pairs of 6‐month‐old porcine lens were retrieved from local slaughterhouse. For each pair, one lens was randomly selected to undergo a localized lesion and the other lens remained intact. The lesion was made on the anterior surface with a steel hexagonal rod (to make the mark easily identifiable) previously immersed in liquid nitrogen for one minute. Both lenses were incubated for 1 h at 20°C in a HEC mixture. Images were acquired with a macroscope and analysed with ImageJ. Calcein‐AM and Ethidium images were used to calculate the area covered by living epithelial cells. Hoescht images allowed to count cell nuclei per unit area. Viable epithelial cell density (vECD) was defined as the number of viable cells per unit area. The lenses were immersed (isotonic saline) inside a black container during the measurements to minimize reflections and refractive phenomena or drying of the samples. Results: The vECD median on the ten pairs was 2840 cells/mm2 [10th‐90th percentiles = 2479–3494] for cryo‐applied lenses versus 3364 cells/mm2 [2919–3739] for healthy lenses (p = 0.002). Conclusions: The triple HEC staining adapted to the whole lens provides an unrivalled measure of pan‐epithelial viability without the need for a tedious capsule dissection step that destroys the lens architecture. Other studies will be necessary to determine its ability to discriminate small variations or type of mortality.