Isolation efficiency of collagenase and EDTA for the culture of corneal endothelial cells

PURPOSE: Tissue engineering of the corneal endothelium, as well as cell therapy, has been proposed as an alternative approach for the treatment of corneal endotheliopathies. These approaches require in vitro amplification of functional corneal endothelial cells (CECs). The goal of this study was to compare two common isolation methods, collagenase A and EDTA (EDTA), and determine whether they influence cell viability, morphology, and barrier function. METHODS: Human eye bank research-grade corneas were used to isolate and cultivate CECs. All donors were more than 40 years old. Two Descemet membranes from the same donor were used separately to compare the collagenase A and EDTA cell isolation methods. The number of isolated cells, cell viability, morphology, and barrier functionality were compared. RESULTS: A higher isolation efficiency of viable CECs and a higher circularity index (endothelial morphology) were obtained using collagenase A. Passage 3 cells presented similar barrier functionalities regardless of the isolation method. CONCLUSIONS: This study showed that isolation of CECs using collagenase A yields higher isolation efficiency than EDTA, delaying the loss of endothelial morphology for early passage cells.