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Preparing media for tissue culture, but not sure how to get it to the right pH? Worried about pH changes during your incubations? DPAG Group publishes recommendations for best practice in controlling and managing pH under culture conditions.

Swietach cell culture image
Cell culture media containing the pH-indicator Phenol Red at different pHs, from acidic (left, yellow) to alkaline (right, pink). Work by Stefania Cannone. Photo credit: KC Park (Swietach Group)

Postdoctoral Research Scientist Johanna Michl and her team in the Swietach Group have used real-time pH measurements of cell culture media and intracellular pH data from live-cell culture conditions to explore the effects of commonly-used buffers. Based on their data, they have proposed guidelines for controlling pH in cell culture systems and improving reproducibility.

Due to its powerful effects on biology, pH is the most fundamental chemical variable that researchers consider when preparing culture media, and one that could, and should, be managed to the highest level of rigour.  However, in the absence of consensus guidelines, various laboratories have adopted strikingly different approaches to controlling pH.

Some methods inadvertently produce pH artefacts that increase noise, compromise reproducibility or even give erroneous inferences.  

As an example, many commonly used media formulations contain 44 mM bicarbonate, which at 5% CO2, equilibrates to the non-physiological pH of 7.7. Media containing non-bicarbonate buffers (e.g. HEPES) may show pH-drift, if prepared incorrectly. Furthermore, medium pH will become disturbed when cells are transferred in and out of CO2 incubators. - Associate Professor Pawel Swietach

In a recent article lead by first author Johanna Michl and published in Communications Biology, the team describe a method for real-time monitoring of extracellular pH and a high-throughput pipeline for measuring intracellular pH under live-cell culture conditions.  The team uses these to show how certain buffering regimes produce pH artefacts, and explain how such errors could be avoided. The paper concludes with guidelines for improving pH control in culture systems.

The full publication Evidence-based guidelines for controlling pH in mammalian live-cell culture systems is available to read here.