Most conventional incubators used in cell culture do not regulate O2 levels, making the headspace O2 concentration ~18%. In contrast, most human tissues are exposed to 2–6% O2 (physioxia) in vivo. Accumulating evidence has shown that such hyperoxic conditions in standard cell culture practices affect a variety of biological processes. In this review, we discuss how supraphysiological O2 levels affect reactive oxygen species (ROS) metabolism and redox homeostasis, gene expression, replicative lifespan, cellular respiration, and mitochondrial dynamics. Furthermore, we present evidence demonstrating how hyperoxic cell culture conditions fail to recapitulate the physiological and pathological behavior of tissues in vivo, including cases of how O2 alters the cellular response to drugs, hormones, and toxicants. We conclude that maintaining physioxia in cell culture is imperative in order to better replicate in vivo-like tissue physiology and pathology, and to avoid artifacts in research involving cell culture.

Mammalian cell culture is a fundamental tool used to study living cells. Presently, the standard protocol for performing cell culture involves the use of commercial media that contain an excess of nutrients. While this reduces the likelihood of cell starvation, it creates non-physiologic culture conditions that have been shown to 're-wire' cellular metabolism. Recently, researchers have developed new media like Plasmax, formulated to approximate the nutrient composition of human blood plasma. Although this represents an improvement in cell culture practice, physiologic media may be vulnerable to nutrient depletion. In this study we directly addressed this concern by measuring the rates of glucose and amino acid depletion from Plasmax in several cancer cell lines (PC-3, LNCaP, MCF-7, SH-SY5Y) over 48 hours. In all cell lines, depletion of glucose from Plasmax was rapid such that, by 48h, cells were hypoglycemic (<2mM glucose). Most amino acids were similarly rapidly depleted to sub-physiological levels by 48h. In contrast, glucose and most amino acids remained within the physiological range at 24h. When the experiment was done at physiological oxygen (5%) versus standard (18%) with LNCaP cells, no effect on glucose or amino acid consumption was observed. Using RNA sequencing, we show that this nutrient depletion is associated with enrichment of starvation responses, apoptotic signalling, and endoplasmic reticulum stress. A shift from glycolytic metabolism to mitochondrial respiration at 5% O2 was also measured using Seahorse analysis. Taken together, these results exemplify the metabolic considerations for Plasmax, highlighting that cell culture in Plasmax requires daily media exchange.

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Mammalian cell culture is widely used for discovery and development. Recently, increasing attention has been paid to the importance of maintaining physiologically-relevant conditions in cell culture. Although oxygen level is a particularly important consideration, it is rarely regulated by experimentalists. The atmospheric O2 levels commonly used in cell culture are significantly higher than those experienced by most mammalian cells in vivo, leaving cells susceptible to oxidative damage, senescence, transformation, and otherwise aberrant physiology. A barrier to incorporating O2 regulation into most cell culture workflows has been the expense of investing in new equipment, as the vast majority of laboratory CO2 incubators do not regulate O2. Here, we describe an inexpensive (<CAD 1000), portable and user-friendly O2/CO2 incubator that can establish and maintain physiological O2, CO2, and temperature values within their physiological ranges. We used an Arduino-based approach to add O2 and CO2 control to a temperature-regulating egg incubator. Our incubator was tested against a commercial laboratory O2/CO2 incubator. Using Presens OxoDish technology, we demonstrate that at a setpoint value of 5% gas-phase incubator O2, media O2 averaged 5.03 (SD = 0.03) with a range of 4.98–5.09%. MCF7, LNCaP and C2C12 cell lines cultured in the incubator displayed normal morphology, proliferation, and viability. Culture for up to one week produced no contamination. Thus, our incubator provides an inexpensive means of maintaining physioxia in routine mammalian cell culture.

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