Cancer cell culture is routinely performed under superphysiologic O2 levels and in media such as Dulbecco’s Modified Eagle Medium (DMEM) with nutrient composition dissimilar to mammalian extracellular fluid. Recently developed cell culture media (e.g., Plasmax, Human Plasma-Like Medium (HPLM)), which are modeled on the metabolite composition of human blood plasma, have been shown to shift key cellular activities in several cancer cell lines. Similar effects have been reported with respect to O2 levels in cell culture. Given these observations, we investigated how media composition and O2 levels affect cellular energy metabolism and mitochondria network structure in MCF7, SaOS2, LNCaP, and Huh7 cells. Cells were cultured in physiologic (5%) or standard (18%) O2 levels, and in physiologic (Plasmax) or standard cell culture media (DMEM). We show that both O2 levels and media composition significantly affect mitochondrial abundance and network structure, concomitantly with changes in cellular bioenergetics. Extracellular acidification rate (ECAR), a proxy for glycolytic activity, was generally higher in cells cultured in DMEM while oxygen consumption rates (OCR) were lower. This effect of media on energy metabolism is an important consideration for the study of cancer drugs that target aspects of energy metabolism, including lactate dehydrogenase activity.

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.

Estradiol (E2) and selective estrogen receptor modulators (SERMs) have broad-ranging cellular effects that include mitochondrial respiration and reactive oxygen species (ROS) metabolism. Many of these effects have been studied using cell culture models. Recent advances have revealed the extent to which cellular metabolism is affected by the culture environment. Cell culture media with metabolite composition similar to blood plasma (e.g. Plasmax, HPLM) alter cellular behaviours including responses to drugs. Similar effects have been observed with respect to O2 levels in cell culture. Given these observations, we set out to determine whether the effects of E2 and SERMs are also influenced by media composition and O2 level during cell culture experiments. We analyzed mitochondrial network characteristics, cellular oxygen consumption rates, and cellular H2O2 production in C2C12 myoblasts growing in physiologic (5%) or standard cell culture (18%) O2 and in physiologic (Plasmax) or standard cell culture (DMEM) media. The cell culture conditions affected all measured parameters under basal conditions and changed how cells responded to E2 or SERMs. These results indicate that the effects of E2 and SERMs on various aspects of cell physiology strongly depends on growth conditions, which in turn emphasizes the need to consider this carefully in cell culture experiments.

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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