Isolation and characterization of bovine Limbal mesenchymal stromal cells for application in ocular toxicity studies

Ocular toxicity assessment is essential to ensure the safety of chemicals and human-use products. Although the Draize test in rabbits has historically been the regulatory standard, it presents significant ethical and scientific limitations. While validated in vitro methods are currently available, most of them do not allow the evaluation of damage reversibility-a key parameter for hazard classification under the United Nations Globally Harmonized System (UN GHS). This study evaluates the utility of an in vitro model based on limbal mesenchymal stromal cells (LMSC) for ocular toxicity testing and the prediction of corneal damage reversibility. LMSC were obtained from slaughterhouse-derived residual tissue, cultured under specific conditions, and briefly exposed to substances classified according to UN GHS. Cell migration was then assessed as an indicator of tissue recovery. This preliminary report presents, to our knowledge, the first application of short-term (5-min) exposure in a scratch assay to evaluate its effect on migration capacity. Unlike previous studies relying on prolonged exposure to inhibitory compounds, this approach better simulates accidental ocular exposure scenarios. While migration was not significantly disrupted under the tested conditions, these results offer valuable insight into the relationship between viability and wound healing and highlight the importance of exposure time and concentration in in vitro ocular toxicity models. Overall, this work establishes a promising foundation for future refinement of alternative methods that aim to predict damage persistence and reversibility more accurately.