Evaluation of ocular irritancy based on cell membrane capacitance on a cruelty-free in vitro corneal model

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 base its prediction on destructive test. In this study, we use a cruelty-free in vitro corneal model (CFreeCM) previously developed by our laboratory, and analysed the changes in cell membrane capacitance using the Standardized Operational Procedures detailed in OECD TG 492 for identifying chemicals not requiring classification and labelling for eye irritation. Cell membranes display the ultrastructure of a natural capacitor due to the dielectric properties of the phospholipid bilayer, meaning that cell capacitance can be related to the cell membrane integrity which could be altered after chemical exposure and might lead to ocular irritation

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. Cell membrane capacitance was evaluated using coupled electrodes connected to a EUCOL U2817A Precission LCR Meter prior to chemical exposure at different frequencies (from 50Hz to 100kHz). Next, 26 chemicals (12 irritants and 14 non-irritants) including liquids and solids were applied in duplicates for 30 minutes or 6 hours respectively. After PBS rinse, CFreeCMs were incubated at 37C for 2 hours for liquids or 18 hours for solids. Finally, cell capacitance was evaluated again and cell viability was assessed using the MTT assay

Results : A prediction model was developed based on changes of cell membrane capacitance and compared to standard classification obtained by the MTT assay. Standard classification according to MTT resulted in 91.6% sensibility (11/12), 64.2% specificity (9/14) and 76.9% accuracy (20/26). Cell capacitance prediction model at 1kHz resulted in 91.6% sensibility (11/12), 78.5% sensibility (11/14) and 88% accuracy (22/26)

Conclusions : Although standard classification based on cell viability is in accordance with OECD TG 492 requirements, classification using cell capacitance resulted in higher specificity and overall accuracy, leading to an optimized non-invasive and non-destructive prediction model of ocular irritancy

This is a 2021 ARVO Annual Meeting abstract.