Controlling Pericellular Oxygen Tension in Cell Culture Reveals Distinct Breast Cancer Responses to Low Oxygen Tensions
Zachary J. Rogers, Thibault Colombani, Saad Khan, Khushbu Bhatt, Alexandra Nukovic, Guanyu Zhou, Benjamin M. Woolston, Cormac T. Taylor, Daniele M. Gilkes, Nikolai Slavov, Sidi A. Bencherif
Abstract
Abstract 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.