Evidence-based Guidelines for Controlling pH in Mammalian Live-Cell Culture Systems

The effect of unstirred medium layers presumably determines the delivery of nutrients/growth factors and the removal of other metabolic waste products such as lactic acid, which can also act to directly and indirectly moderate environmental variation.

“The effect of unstirred medium layers also presumably determines the delivery of nutrients/growth factors and the removal of other metabolic waste products (e.g., lactic acid), which can also act to directly and indirectly moderate environmental variation (Michl et al., 2019).”

The majority of research papers rely on nominal set points and fail to directly verify and report environmental parameters.

“recent assessments show that the majority of research papers rely on nominal set points and fail to directly verify and report environmental parameters (Al-Ani et al., 2018; Michl et al., 2019; Klein et al., 2021b).”

Cellular growth of three colorectal cell-lines (NCI-H747, DLD1, Caco2) was optimal at pH 7.4, but when medium pH deviated from 7.4 by >0.3 units all three cell lines exhibited reduced rates of proliferation.

“Michl and others (2019) showed that cellular growth of three colorectal cell-lines (NCI-H747, DLD1, Caco2) was optimal at pH 7.4, but when medium pH deviated from 7.4 by > 0.3 units all three cell lines exhibited reduced rates of proliferation.”

Titration of acids and bases (including HCl, NaOH, and NVBs) introduces osmolytes (Na+, Cl−) to cell medium and can result in substantial changes to medium osmolarity by >10%.

“The titration of acids and bases (including HCl, NaOH, and NVBs) introduces osmolytes (Na+, Cl−) to cell medium and can result in substantial changes to medium osmolarity by > 10% (Michl et al., 2019).”

In batch culture set-ups, researchers often select culture medium that contains a pH indicator dye such as Phenol Red to guide the renewal of cell medium, but medium color changes assessed "by eye" can lead to undetected pH declines.

“In batch culture set-ups, researchers often select culture medium that contains a pH indicator dye [e.g., Phenol Red (PhR)] to guide the renewal of cell medium, but medium color changes assessed “by eye” can lead to undetected pH declines (Michl et al., 2019).”

The initial stability of medium pH is typically achieved by mimicking the physiologically relevant CO2/HCO3− buffering system.

“The initial stability of medium pH is typically achieved by mimicking the physiologically relevant CO2/HCO3 − buffering system (Michl et al., 2019).”

Exogenous buffer approaches can, in some cases, promote unpredictable changes in pH and introduce confounding artifacts.

“such approaches can, in some cases, promote unpredictable changes in pH and introduce confounding artifacts (Michl et al., 2019, see below).”

Perturbations to dissolved O2 levels in culture medium can induce cells to switch away from oxidative phosphorylation towards anaerobic glycolysis, leading to large accumulations of lactic acid that force medium acidification.

“For instance, perturbations to dissolved O2 levels in culture medium can induce cells to switch away from oxidative phosphorylation towards anaerobic glycolysis (Wolff et al., 1993), leading to large accumulations of lactic acid that force medium acidification (Michl et al., 2019).”

The CO2/HCO3− buffer system is the primary physiological buffering system in mammalian fluids.

“Although the CO2/HCO3 − buffer system is indeed the primary physiological buffering system in mammalian fluids (Boron, 2004; Michl et al., 2019)”

Another post-publication analysis reported that less than half of studies published in Cancer Research and Nature in the third quarter of 2017 described the brand of medium, and only one-tenth declared the medium-buffering regime.

“Another post-publication analysis reported that less than half of studies published in Cancer Research and Nature in the third quarter of 2017 described the brand of medium, and only one-tenth declared the medium-buffering regime (Michl et al., 2019).”

NVB addition may not fully prevent pH declines in standard batch cultures and may lead to unexpected pH changes when interacting with the HCO3−/CO2 buffering system, although predictable pH levels can be obtained when appropriate protocols are used.

“Recent assessments also shows that NVB addition may not fully prevent pH declines in standard batch cultures and may lead to unexpected pH changes when interacting with the HCO3 −/CO2 buffering system, although predictable pH levels can be obtained when appropriate protocols are used (see, Michl et al., 2019).”