Optimized Protocol for Isolation and Culture of Primary Human Corneal Epithelial Cells.

Purpose: To establish a reliable method for isolating and culturing high-purity primary human corneal epithelial cells (HCECs) for ophthalmic drug testing. Methods: We present a detailed, step-by-step protocol for the efficient isolation and culture of primary HCECs. This protocol includes the characterization of HCEC morphological responses to varying Ca2+ concentrations in the culture medium. Additionally, immunofluorescence staining with well-established markers is used to identify and confirm the cell types in vitro. A Ca2+ assay is performed to validate the functionality of the cultured primary HCECs. Results: By following the procedures detailed in this protocol, high-purity primary HCECs with strong proliferative capacity and preserved morphological integrity are obtained. Immunofluorescence staining confirms the presence of both limbal stem cells and differentiated corneal epithelial cells in vitro. Additionally, the functional assay demonstrates that the cultured primary HCECs retain the ability to respond to external Ca²⁺ stimuli. Conclusions: This optimized protocol enhances the efficiency and reliability of primary HCECs isolation and culture, enabling the development of a robust in vitro model for studying the mechanisms of ocular diseases. Translational Relevance: Successfully cultured primary HCECs in vitro bridges the gap between laboratory findings and clinical applications, facilitating advancements in therapeutic development.