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Sangita P. Patel, Brayan Calle Gonzalez, Nataliia Paone, Christian Mueller, Jamie C. Floss, Maria E. Sousa, Michael Y. Shi; Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures. Trans. Vis. Sci. Tech. 2022;11(2):33. doi: https://doi.org/10.1167/tvst.11.2.33.
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Primary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O2 concentrations [O2] than what is normally present in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O2] (2.5%; [O2]2.5) and room air ([O2]A).
Primary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O2]2.5 and [O2]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 [O2] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates.
HCEnCs grown at [O2]A and [O2]2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O2]2.5 than at [O2]A. HIF-1α was undetectable. Cells displayed greater viability at [O2]2.5 than at [O2]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.
Primary HCEnCs show unique metabolic characteristics at physiologic [O2]. The effect of [O2] for optimization of HCEnC culture conditions should be considered.
With the advance of cell-based therapeutics for corneal endothelial diseases, [O2] should be considered an important variable in the optimization of HCEnC culture conditions.
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