Dissolved oxygen concentration in the medium during cell culture: Defects and improvements

In vitro cell culture has provided a useful model to study the effects of oxygen on cellular behavior. However, it remains unknown whether the in vitro operations themselves affect the medium oxygen levels and the living states of cells. In addition, a prevailing controversy is whether reactive oxygen species (ROS) production is induced by continuous hypoxia or reoxygenation. In this study, we have measured the effects of different types of cell culture containers and the oxygen environment where medium replacement takes place on the actual oxygen tension in the medium. We found that the deviations of oxygen concentrations in the medium are much greater in 25‐cm2 flasks than in 24‐well plates and 35‐mm dishes. The dissolved oxygen concentrations in the medium were increased after medium replacement in normoxia, but remained unchanged in glove boxes in which the oxygen tension remained at a low level (11.4, 5.7, and 0.5% O2). We also found that medium replacement in normoxia increased the number of ROS‐positive cells and reduced the cell viability; meanwhile, medium replacement in a glove box did not produce the above effects. Therefore, we conclude that the use of 25‐cm2 flasks should be avoided and demonstrate that continuous hypoxia does not produce ROS, whereas the reoxygenation that occurs during the harvesting of cells leads to ROS and induces cell death.