How Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions

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.