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-1 alpha (hypoxia-inducible factor-1 alpha) 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-1 alpha 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.