Optimization of culture conditions for the human corneal epithelial cell line IM-HCEpiC and evaluation of its extracellular vesicles in repair

Abstract

Abstract Corneal epithelial defects are a major cause of vision loss, highlighting the need for reliable in vitro models to study ocular surface pathophysiology and for therapeutic development. In this study, we establish a simplified and reproducible protocol for culturing the human corneal epithelial cell line IM-HCEpiC under standardized culture conditions, avoiding coated flasks and proprietary media of unknown composition. We compared proliferation rates across different seeding densities and culture media formulations, identifying DMEM/F12 supplemented with fetal bovine serum as the optimal compositionally defined medium, while collagen coating does not significantly improve growth. Under these conditions, IM-HCEpiC cells expressed corneal epithelial markers (CK3, CK12, ZO-1, E-cadherin, and Pax-6) and displayed the expected response to oxidative stress and antioxidant treatment, supporting the preservation of key epithelial characteristics. Using this optimized culture system, extracellular vesicles (EVs) were isolated from IM-HCEpiC secretomes, characterized, and functionally evaluated. IM-HCEpiC-derived EVs promote corneal wound closure and increased Ki67 expression. Therefore, this study establishes a simplified, cost-effective, and reproducible culture protocol for IM-HCEpiC cells while preserving key corneal epithelial characteristics, supporting their use in corneal epithelial biology and EV-based research.