Human corneal epithelial cells harvested from advanced surface ablation (ASA): An optimized in vitro culture protocol

Abstract

Purpose: Corneal epithelial cells obtained from advanced surface ablation (ASA) surgery provide a valuable resource for in vitro models of ocular surface diseases. The aim of this study is to enhance culture conditions and characterize the functionality of EpiASA cells in culture, focusing on their ability to keep the distinctive prop- erties of corneal epithelial cells. Methods: EpiASA samples from 51 patients were included in the study. Two different collection media were tested, and their effect on sample preservation and initial viability was evaluated. Then, cells were disaggregated and cultured using different strategies to increase cell viability, which was measured by AlamarBlue assay. Once the optimized conditions were established, cells were cultured and passaged, and structural and functional characterization of native tissue, primary cultures, and first-passage cultures was performed using atomic force microscopy (AFM), qPCR, and immunofluorescence stainings. Results: The addition of trehalose to the basal collection medium increased EpiASA initial viability. Culture surface coating with type I collagen, along with the supplementation of culture medium with hydrocortisone, significantly increased cell viability. On the contrary, co-cultures with different ocular cell lines, or the use of human serum, did not provide a sustained benefit. Further low-concentration trehalose supplementation of EpiASA cultures enhanced monolayer formation and allowed subculturing. AFM and immunofluorescence confirmed that passage 1 EpiASA cells retained corneal epithelial characteristics, including well-organized microvilli and uniform expression of barrier and epithelial markers. Conclusion: This research provides an optimized protocol (EpiKeraMAX) for using EpiASA samples for in vitro studies of human corneal cells