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.
(© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)