Blue Light-Based Method to Induce Oxidative Stress on Rabbit Corneal Epithelial (RCE) Cells: Development and Validation

Daily exposure to blue light emitted by digital devices has raised concerns about oxidative stress-mediated damage to the ocular surface. Despite growing interest, validated in vitro models to study blue light-induced oxidative stress in corneal epithelial cells remain limited. A reproducible in vitro method was developed using rabbit corneal epithelial (RCE) cells exposed to blue LED light (405 nm). Irradiation parameters were optimized to induce oxidative stress without causing overt cytotoxicity. Cellular viability, intracellular ROS production, and mitochondrial oxidative stress were assessed. The model was validated using reference antioxidants (ascorbic acid and oleuropein), oleuropein formulated in a drug-in-cyclodextrin-in-liposome system (OLE-DCL), and two commercial ophthalmic formulations applied before or after irradiation. Blue light irradiation at 4.57 W/m2 for 30 min significantly increased intracellular and mitochondrial ROS levels while preserving cell viability, indicating sublethal photo-oxidative stress. Ascorbic acid effectively suppressed ROS generation, whereas free oleuropein showed reduced efficacy, likely due to photosensitivity. OLE-DCL significantly enhanced antioxidant activity under irradiation. The model also discriminated between protective and restorative treatment strategies. This study establishes a validated in vitro blue light-induced oxidative stress model for corneal epithelial cells, suitable for screening antioxidant compounds, formulations, and application strategies relevant to ocular surface protection.