DOI: 10.5061/dryad.ngf1vhhwq
title: 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

description: 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. 
description: 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 CO<sub>2</sub>,
 N<sub>2</sub>, and O<sub>2</sub> 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: CO<sub>2</sub> gas was
 delivered to lower pH and N<sub>2</sub> gas was used to
 increase pH. The use of this method decreased the pH by reflecting the
 changes in CO<sub>2</sub> equilibria. A one-sided control loop
 (pure O<sub>2</sub>) was used to maintain
 O<sub>2</sub> levels at ~90% air saturation in all
 treatments.  Environmental data (dissolved oxygen and pH) were constantly
 monitored with the integrated pH and dO<sub>2</sub> 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. 
description: 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.