A proof-of-concept study on the use of Optisol-GS for short-term cold preservation of bovine corneas applied to the BCOP assay.

The Bovine Corneal Opacity and Permeability (BCOP) assay is a widely adopted non-animal method for identifying substances capable of causing severe eye damage. However, its implementation is limited by the requirement for freshly excised bovine corneas, collected and tested within a narrow post-mortem window. This proof-of-concept study evaluated whether short-term hypothermic preservation in Optisol-GS, a clinically validated corneal storage medium, could extend the use bovine corneas for BCOP assay. Corneas were preserved for up to 14 days and assessed for baseline opacity, control performance, IVIS classification of selected OECD proficiency chemicals, and histological integrity. Optisol-GS maintained corneal suitability for up to seven days, whereas HBSS and EMEM retained tissue acceptability only within the first 24 h. Preserved corneas consistently met BCOP validity criteria, yielding stable negative and positive control responses and expected IVIS classifications for all OECD proficiency substances tested. Histological analyses (HE, PAS, Masson) confirmed preservation of key epithelial, stromal, and endothelial structures, as well as depth-dependent injury patterns consistent with chemical severity. These findings indicate that Optisol-GS effectively extends the operational window for BCOP without compromising assay performance, suggesting a practical solution to logistical constraints associated with fresh tissue availability and supporting broader implementation of validated New Approach Methodologies in ocular toxicology.