Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures

PURPOSE: Primary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O(2) concentrations [O(2)] than what is normally present in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O(2)] (2.5%; [O(2)](2.5)) and room air ([O(2)](A)). METHODS: Primary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O(2)](2.5) and [O(2)](A). Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O(2)] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates. RESULTS: HCEnCs grown at [O(2)](A) and [O(2)](2.5) displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O(2)](2.5) than at [O(2)](A). HIF-1α was undetectable. Cells displayed greater viability at [O(2)](2.5) than at [O(2)](A). HCEnCs showed significantly greater proton leak (P < 0.01), nonmitochondrial oxygen consumption (P < 0.01), and spare capacity (P < 0.05) for oxygen consumption rates, and greater basal glycolysis (P < 0.05) with a decreased glycolytic reserve capacity (P < 0.05) for extracellular acidification rates. CONCLUSIONS: Primary HCEnCs show unique metabolic characteristics at physiologic [O(2)]. The effect of [O(2)] for optimization of HCEnC culture conditions should be considered. TRANSLATIONAL RELEVANCE: With the advance of cell-based therapeutics for corneal endothelial diseases, [O(2)] should be considered an important variable in the optimization of HCEnC culture conditions.