Isolation of Corneal and Limbal Cells From Human Donor Cornea

Objective: Isolation of corneal cells while preserving their specific phenotypes is crucial for regenerative medicine applications. Here, we aimed to evaluate corneal cell isolation techniques from human corneal tissue. Methods: We isolated corneal cells and limbal stem cells from human donor corneas obtained from the eye bank by employing both enzymatic and explant cell culture approaches and monitored the morphological characteristics of the isolated cells. Results: Explant culture demonstrated suitability for the isolation of corneal endothelial and epithelial cells, while both explant and enzymatic culture processes yielded successful results for limbal and corneal stromal tissues. The optimal conditions for enzymatic culture were determined to be 2 mg/mL enzyme solution for 30 minutes at 37 °C. On the other hand, the feasibility of explant cultures was increased by re-transferring and culturing tissue specimens to isolate an enlarged quantity of cells in an extended culture periods. However, this approach resulted in morphological changes in the isolated cells. The earliest passage at which isolated primary cells (keratocyte and limbal stem cells) could be immortalized was Passage 1, enabling subculturing and cell stock creation. On the other hand, isolated primary limbal stem cells demonstrated the capacity to differentiate into corneal epithelial and keratocyte cells. Conclusion: This study provides a comprehensive and detailed presentation that will serve as an informative resource in the literature for corneal cell culture, corneal tissue engineering, and drug delivery studies.