Most mammalian tissue cells experience oxygen partial pressures in vivo equivalent to 1–6% O2 (i.e., physioxia). In standard cell culture, however, headspace O2 levels are usually not actively regulated and under these conditions are ~18%. This drives hyperoxia in cell culture media that can affect a wide variety of cellular activities and may compromise the ability of in vitro models to reproduce in vivo biology. Here, we review and discuss some specific O2-consuming organelles and enzymes, including mitochondria, NADPH oxidases, the transplasma membrane redox system, nitric oxide synthases, xanthine oxidase, and monoamine oxidase with respect to their sensitivities to O2 levels. Many of these produce reactive oxygen and/or nitrogen species (ROS/RNS) as either primary end products or byproducts and are acutely sensitive to O2 levels in the range from 1% to 18%. Interestingly, many of them are also transcriptional targets of hypoxia-inducible factors (HIFs) and chronic cell growth at physioxia versus 18% O2 may alter their expression. Aquaporins, which facilitate hydrogen peroxide diffusion into and out of cells, are also regulated by HIFs, indicating that O2 levels may affect intercellular communication via hydrogen peroxide. The O2 sensitivities of these important activities emphasize the importance of maintaining physioxia in culture.

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Resveratrol is a plant-derived polyphenol that has been widely studied for its putative health promoting effects. Many of those studies have been conducted in cell culture, in supra-physiological levels of oxygen and glucose. Resveratrol interacts with reactive oxygen species (ROS) as an antioxidant or pro-oxidant. Resveratrol affects the expression and activities of ROS-producing enzymes and organelles. It is therefore important to consider how cell culture conditions might determine the effects of resveratrol on cultured cells. We determined the effects of resveratrol on cell growth, hydrogen peroxide production, and mitochondrial network characteristics in C2C12 mouse myoblasts and PC3 human prostate cancer cells under conditions of physiological (5%) and supra-physiological (18%) oxygen, and normo- (5 mM) and hyper-glycemia (25 mM). Interestingly, most effects of resveratrol on the parameters measured here were dependent upon prevailing oxygen and glucose levels during the experiment. Many of the effects of resveratrol on cell growth, hydrogen peroxide production, and mitochondrial network characteristics that were seen in 25 mM glucose and/or 18% oxygen were absent under the physiologically relevant conditions of 5 mM glucose with 5% oxygen. These findings emphasize the importance of using physiologically meaningful starting conditions for cell-culture experiments with resveratrol and indeed any manipulation affecting ROS metabolism and mitochondria.

Cancer stem cells (CSCs) are a small population of resistant cells inhabiting the tumors. Although comprising only nearly 3% of the tumor mass, these cells were demonstrated to orchestrate tumorigenesis and differentiation, underlie tumors’ heterogeneity and mediate therapy resistance and tumor relapse. Here we show that CSCs may be formed by dedifferentiation of terminally differentiated tumor cells under stress conditions. Using a elegant co-culture cellular system, we were able to prove that nutrients and oxygen deprivation activated non-malignant stromal fibroblasts, which in turn established with tumor cells a paracrine loop mediated by Interleukine-6 (IL-6), Activin-A and Granulocyte colony-stimulating factor (G-CSF), that drove subsequent tumor formation and cellular dedifferentiation. However, by scavenging these cytokines from the media and/or blocking exosomes’ mediated communication it was possible to abrogate dedifferentiation thus turning these mechanisms into potential therapeutic targets against cancer progression.