Optimizing oxygen in cell culture through a pericellular oxygen regulating incubator

The vast majority of cell culture is done in incubators that do not regulate oxygen (O 2 ) levels. Increasingly, however, researchers are recognizing the importance of culturing cells at physiological oxygen levels, which are typically 2-6% O 2 compared to 18-19% when not regulated. Culturing at levels as low as 1% O 2 is also common, often in the context of cancer biology where hypoxia is a feature of many solid tumours. O 2 -regulating incubators are sometimes employed by researchers, but these incubators regulate oxygen in the gas phase. However, cellular oxygen consumption leads to oxygen depletion in the liquid phase of media immediately adjacent to the cells. Consequently, these pericellular O 2 levels are much lower than intended and reported by researchers. In fact, it is likely that most hypoxia research is performed under nearly anoxic conditions. The extent of oxygen depletion in media is dependent on cell density, cell type, and height of the media column among other variables; these are virtually never reported in researcher articles, greatly limiting reproducibility. This needs to be rectified to optimize the cell culture model. To this end, we have developed a cell culture incubator that regulates O 2 in the liquid phase, rather than headspace gas. This innovation allows for a far better standardization of the oxygen concentration experienced by cells. We will show that this incubator can regulate pericellular oxygen at different set points and at different seeding densities. In the future, this technology is vital for physiological cell culture and hypoxia research. Funding from Natural Science and Engineering Council of Canada Discovery Grant to JAS This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.