Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures

Abstract

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.

Selective effects of estradiol on human corneal endothelial cells

“…Western blots were performed similarly to published protocols [22]. Protein was isolated from confluent PC3 and MCF7 cultures.…”

“…For half of the wells, the incubation in minimal medium included addition of 10 nM E2 and the other half served as control without E2. The Seahorse XFe24 sensor cartridges and drugs were prepared according to manufacturer protocols as previously described by our laboratory [22]. The assay medium was Seahorse XF Dulbecco's modified Eagle medium, with 1.2 mM glutamine, 7.0 mM glucose, and 0.45 mM pyruvate.…”

“…We evaluated the effects of E2 and G1 on HCEnC growth. The HCEnCs in these experiments were cultured under physiological O 2 conditions [28] with 2.5% O 2 ([O 2 ] 2.5 ) and under the stress of chronic high oxygen conditions in a room air culture incubator ([O 2 ] A ) [22]. These two conditions were chosen in order to model the oxygen stress seen in FECD [29].…”

Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids

“…The authors concluded that evaluating cancer cells under physioxia could more closely recapitulate their physiopathologic status in the in vivo microenvironment. Although IEO studies are generally performed at 18-21% oxygen, several have examined the benefits of growing other ex vivo models in physioxia ( 34 – 37 ). For example, kidney organoids cultured in physiological hypoxia (7% oxygen) instead of 21% oxygen showed enhanced sprouting and interconnectivity while maintaining renal cell types and their spatial organization ( 33 ).…”

“…For example, kidney organoids cultured in physiological hypoxia (7% oxygen) instead of 21% oxygen showed enhanced sprouting and interconnectivity while maintaining renal cell types and their spatial organization ( 33 ). Primary human corneal endothelial cells can successfully be cultured at 2.5% oxygen resembling in vivo corneal aqueous environment containing 2.8% oxygen ( 37 ). Compared to room air (∼21% oxygen), corneal endothelial cells cultured at physiological hypoxia showed considerable differences in cell metabolism, viability, oxygen-consuming reactions, and glycolytic metabolism.…”