Controlling Pericellular Oxygen Tension in Cell Culture Reveals Distinct Breast Cancer Responses to Low Oxygen Tensions.

In oxygen (O 2 )-controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O 2 tension experienced by cells (i.e., pericellular O 2 ). However, it is discovered that physioxic (5% O 2 ) and hypoxic (1% O 2 ) setpoints regularly induce anoxic (0% O 2 ) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O 2 consumption is the driving factor. RNA-seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to cellular O 2 consumption. A reaction-diffusion model is developed to predict pericellular O 2 tension a priori, demonstrating that the effect of cellular O 2 consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O 2 tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia-inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non-monotonically to low O 2 , suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O 2 tension in cell culture incubators is insufficient to regulate O 2 in cell culture, thus introducing the concept of pericellular O 2 -controlled cell culture.