Cruelty-free corneal damage assay based on automatic high-throughput cell capacitance

Abstract

Purpose : Animal testing remains as the gold standard for the evaluation of potential damages on the ocular surface of topical products. Although alternative methods are currently available via in vitro reconstructed epithelia, these methods rely on animal products such as fetal bovine serum or co-cultures with mouse fibroblast and base its prediction on destructive test. In this study we introduce the use of a true cruelty-free cornea model (CFreeCM), and analyzed the changes in the accumulation of electric charge on the lipid membranes, after alternating potential pulses, for its use as a non-destructive test for corneal damage prediction

Methods : CFreeCMs were prepared from limbal cells isolated from human normal corneoescleral rims discarded after corneal keratoplasty using animal component-free culture media. Limbal cells were cultured on 1.12cm² Transwell insert and differentiated for 7 days under air-lift conditions. CFreeCMs were exposed to increasing concentrations (from 0 mg/mL to 6 mg/mL) of SDS for 30 minutes. Then, models were washed in PBS and cell capacitance (Cp) was immediately measured on an Electrochemical Impedance Spectroscopy at different frequencies (from 100 Hz to 500 kHz). Subsequently, cell viability was evaluated using the MTT assay. The correlation between Cp and cell viability measures was analyzed by regression analysis using Igor Pro v6.3.7

Results : CFreeCMs present normal corneal phenotype characterized by an undifferentiated basal cell layer and terminally differentiated wing and squamous cell layer. In this model, corneal damage assessed using low frequency Cp values (100 Hz to 1 kHz) resulted in non-linear data sets that could not be appropriately fitted to cell viability measures. Higher frequencies (300 kHz to 500 kHz) resulted in invariant Cp values in relation to cell viability measures. Intermediate frequencies (1 kHz to 100 kHz) resulted in correlation coefficients (R²) greater than 0.9 in a linear regression plot between Cp and cell viability

Conclusions : A true CFreeCM could be developed using human normal limbal cells. By using appropriate frequencies of voltage pulses, cell viability could be predicted in a non-destructive assay. If confirmed by future studies, the use of Cp would offer a novel non-destructive test method for the evaluation of corneal damage, making regulatory studies that lack a cruelty-free alternative method, such as repeated dose toxicity, a reality

This is a 2020 ARVO Annual Meeting abstract.