Key donor corneal factors governing successful expansion of human corneal endothelial cells

Primary human corneal endothelial cell (CEC) culture is a key step toward developing cell-based therapies for corneal endothelial diseases, yet its success remains highly variable, particularly when using tissues from older donors. This study investigated donor-related corneal factors associated with successful CEC expansion in vitro. A total of 310 human donor corneas preserved in organ culture were classified into three age groups: ≥50 years (n = 285), 30-49 years (n = 12), and ≤ 29 years (n = 13). Culture success was defined by typical endothelial morphology and an endothelial cell density (ECD) ≥ 1500 cells/mm² at passage 3. Success rates declined significantly with donor age, reaching 92% in young donors, 42% in intermediate donors, and 12% in older donors (p < 0.0001). Among corneas from older donors, higher initial ECD and shorter organ culture duration were independently associated with successful expansion, whereas post-mortem time (≤ 25 h) showed no significant effect. Unsuccessful cultures most frequently displayed cellular hypertrophy or mesenchymal-like morphology. Applying selection thresholds of initial ECD ≥ 1700 cells/mm² and organ culture duration ≤ 15 days increased the success rate in older donors to approximately 25%. These findings identify key donor selection criteria that improve primary CEC culture outcomes and provide practical guidance for optimizing tissue use in the development of corneal endothelial cell-based therapies.