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.

Automated summary

This study evaluated the growth and metabolism of corneal endothelial cells cultured at different oxygen concentrations. The results showed that corneal cells cultured at physiological oxygen concentrations exhibited unique metabolic characteristics, while cells cultured in room air had poorer growth. This is important for optimizing corneal cell culture conditions.