Oxygen control in cell culture - Your cells may not be experiencing what you think!

Oxygen (O₂)-controlled cell culture has been pivotal in studying mammalian mechanisms of O₂ sensing, regulation, and utilization. We posit, however, that O₂-controlled cell culture is paradoxically not controlling O₂. There is overwhelming evidence that the pericellular O₂ is lower than the surrounding gas phase due to cellular O₂ consumption. Standard hypoxic cell culture is at high risk of inducing pericellular anoxia. We discuss the implications of poor O₂ control for cellular O₂ regulation mechanisms, bioenergetics, and redox signaling. We also highlight the evidence of frequent under-oxygenation in standard (i.e., normoxic) cell culture. This issue has been largely overlooked because strategies to control pericellular O₂ have been lacking. Here, we propose a framework to control pericellular O₂ based on our recent investigation into the nature of the gas/pericellular O₂ gradient. Implementing this framework into standard practice will unlock quantitative O₂ control in vitro, improving our ability to understand the role of O₂ in biology.