A gas-only bioreactor system maintains stable culture environments and reveals that moderate pH deviations trigger transcriptome-wide responses in human cells cultured in physioxia and physiological buffers

Although pH instability is emerging as a potential driver of changes in cell physiology, pH is still poorly controlled during cell culture and in vitro experiments. Standard procedures include the use of chemicals usually not present in the primary physiological buffering system (CO2/HCO3-), such as acids and bases, to manipulate pH levels. This, however, leads to artifacts that potentially affect scientists’ findings. Here, we propose a novel method for controlling pH levels by relying only on the physiological buffering system. pH was manipulated in a repurposed commercial bioreactor set-up, using a two-sided control loop of CO2 and N2 gas in NaHCO3--buffered medium. This method produces optimal and stable dO2 profiles and tightly maintains pH levels. With this procedure, we analyzed the effects of different pH levels (6.8, 7.0, 7.2, and 7.4) on the performance and transcriptome of the human GM12878 cell line over a 72-hours experiment. Our results showed that inflammation and negative cellular proliferation are among the signatures activated at low pH. This further highlights the importance of a thorough pH control during cell culture.