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.

Fuchs endothelial corneal dystrophy (FECD) results from genetic and environmental factors triggering mitochondrial and oxidative stress in corneal endothelial cells (CEnCs) leading to CEnC death and corneal opacification. FECD is more common in women than men, but the basis for this observation is unknown. Because FECD is commonly diagnosed around the time of the menopausal transition in women when estrogen levels decrease precipitously, we studied the effects of the potent estrogen,17-β estradiol (E2) on growth, oxidative stress, and metabolism in primary cultures of human CEnCs (HCEnCs) under conditions of physiologic 2.5% O2 ([O2]2.5) and under hyperoxic stress ([O2]A: room air + 5% CO2). We hypothesized that E2 would counter the stresses of the hyperoxic environment in HCEnCs. HCEnCs were treated ±10 nM E2 for 7-10 days at [O2]2.5 and [O2]A followed by measurements of cell density, viability, reactive oxygen species (ROS), mitochondrial morphology, oxidative DNA damage, ATP levels, mitochondrial respiration (O2 consumption rate [OCR]), and glycolysis (extracellular acidification rate [ECAR]). There were no significant changes in HCEnC density, viability, ROS levels, oxidative DNA damage, OCR, and ECAR in response to E2 under either O2 condition. We found that E2 disrupted mitochondrial morphology in HCEnCs from female donors but not male donors at the [O2]A condition. ATP levels were significantly higher at [O2]2.5 compared to [O2]A in HCEnCs from female donors only, but were not affected by E2. Our findings demonstrate the overall resilience of primary HCEnCs against hyperoxic stress. The selective detrimental effects of hyperoxia and estradiol on HCEnCs from female but not male donors suggests mechanisms of toxicity based upon cell-sex in addition to hormonal environment.