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. 

The effects of four pH levels (6.8, 7.0, 7.2, and 7.4) on the transcriptome and cell performance of the human lymphoblastoid cells (GM12878 cell line) were assessed using a repurposed bioreactor. A DASBox® Mini Bioreactor system controlled by the DASware® Software Suite was used to deliver CO₂, N₂, and O₂ gas to four bioreactor units. We did so, by reprogramming the bioreactor to use a two-sided feedback loop. Briefly, deviations in pH above or below the setpoint would trigger the addition of gas as follows: CO₂ gas was delivered to lower pH and N₂ gas was used to increase pH. The use of this method decreased the pH by reflecting the changes in CO₂ equilibria. A one-sided control loop (pure O₂) was used to maintain O₂ levels at ~90% air saturation in all treatments.  Environmental data (dissolved oxygen and pH) were constantly monitored with the integrated pH and dO₂ sensors. Cell samples were collected at 24, 48, and 72 hours from each of the four replicate vessels within each pH treatment (i.e., n =16) and stored for transcriptomic and cell performance analyses. 

Raw environmental data (dissolved oxygen and pH) are reported at 30-minute intervals for the four pH treatments (6.8, 7.0, 7.2, and 7.4) over the 72-hours experiment. Dissolved oxygen is reported in % air saturation. Data are organized in columns indicating the Timepoint (i.e., the sampling time), Trial number (i.e., the technical replicate number), and pH level (pH 6.8, pH 7.0, pH 7.2, pH 7.4). Cell performance data about cell viability (%) and cell count ml-1 are reported for the four pH treatments (6.8, 7.0, 7.2, and 7.4) over the 72-hours experiment. Columns “Timepoint”, “Technical replicate”, “Bioreactor Unit”, and “Trial number” indicate the sampling time, the technical replicate number, the bioreactor number (from 1 to 4), and the number of the four consecutive experiments conducted to obtain sufficient replication (from 1 to 4), respectively. Missing values represent unavailable data.