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Abstract
A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid–base chemistry. Although awareness of the importance of pH is fostered early in the training of researchers, there are no consensus guidelines for best practice in managing pH in cell cultures, and reporting standards relating to pH are typically inadequate. Furthermore, many laboratories adopt bespoke approaches to controlling pH, some of which inadvertently produce artefacts that increase noise, compromise reproducibility or lead to the misinterpretation of data. Here, we use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, including physiological CO2/HCO3− and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. To improve data reproducibility, we propose guidelines for controlling pH in culture systems.
Highlights
A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid–base chemistry
We relate HCO3− and CO2 with pH, show how the system responds to changes in its components and demonstrate how the equilibrium is affected by non-volatile buffers (NVBs) added to augment buffering capacity
We propose guidelines for good practice in controlling pH in culture systems
Summary
A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid–base chemistry. We use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, including physiological CO2/HCO3− and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. The combination of CO2 (an acidic gas) with HCO3− (a base) produces quantitatively the most important buffer in extracellular body fluids In culture systems, this so-called carbonic buffer is stabilized by adding an amount of HCO3− salt to media and enriching the incubator's atmosphere with CO2. We propose guidelines for good practice in controlling pH in culture systems
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