Evidence-based guidelines for controlling pH in mammalian live-cell culture systems

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.

Keywords: Cancer microenvironment; Cell biology; Cell culture; Cytological techniques.

Fig. 1

Measuring and setting medium pH under incubation. a Absorbance spectrum of Phenol Red (PhR) in Dulbecco’s modified Eagle’s medium (DMEM) (D7777) with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin (PS), 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) plus 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), and titrated (5 M HCl or 4 M NaOH) to the indicated pH. Arrows indicate wavelengths for optimal ratiometric analysis. b pH dependence of 560/430 nm ratio, fitted to curve: pH = 8.35 + log((10.9 – ratio))/(ratio – 0.0392)). c Controlling equilibrium pH by varying pCO₂ and [HCO₃⁻] in DMEM (D7777) supplemented with 10% FBS and 1% PS. Dashed line plots Eq. 1. Continuous line is best fit to Eq. 3 (corrected version of Eq. 1), which accounts for buffering by serum (best fit: 1.11 mM pH⁻¹). Inset replots the data at low [HCO₃]. d Empirical determination of intrinsic buffering capacity of DMEM (D7777; 10% FBS/1% PS, 25 mM glucose) nominally lacking buffers; titration with either HCl or NaOH. Inverse of slope  provides an estimate of buffering due to serum proteins and media salts. All measurements were repeated three times (three technical replicates each). Data are shown as mean ± SEM

Fig. 2

The control and stability of pH in CO₂/HCO₃⁻-buffered medium. a Effect of increasing [lactic acid] or [lactate] on equilibrium pH of medium (DMEM D5030) with 22 mM NaHCO₃ (10% foetal bovine serum (FBS), 1% penicillin–streptomycin (PS)) placed in 5% CO₂. b Effect of metabolic lactic acid production by DLD1 cells (seeding density 4,000 cells per well, growth area 0.32 cm² per well) on the pH of medium (DMEM D5030; 10% FBS,  1% PS) containing 0–25 mM glucose (osmotically compensated with NaCl). Error bars omitted for clarity. c Effect of varying starting glucose concentration on net glucose uptake and lactate production probed on the seventh day of incubation. 90% of glucose is metabolized to lactate. d Relationship between lactate production (measured by biochemical assay) and total acid production (calculated from the pH change and buffering capacity). Slope of 1.0 indicates that medium acidification is due to lactic acid production. e Cell growth of three colorectal cancer cell lines (seeding density 4,000 cells per well, growth area 0.32 cm² per well) measured from protein biomass (sulforhodamine B (SRB) assay) after 6 days of incubation in DMEM (D7777; 10% FBS, 1% PS, 25 mM glucose) over a range of starting pH attained by varying [HCO₃⁻] at constant 5% CO₂. Data are normalized to the optimum pH derived by best fit to biphasic curve. Optimal growth is near the physiological pH of 7.4. f Effect of varying pCO₂ on medium pH, mimicking the withdrawal of medium from under CO₂ incubation. All experiments were repeated three times (three technical replicates each). Data are shown as mean ± SEM

Fig. 3

pH dynamics in media prepared with non-volatile buffers without consideration of the CO₂-HCO₃⁻ equilibrium. a Medium  (DMEM D7777) supplemented with non-volatile buffer (HEPES, PIPES or MES; 20 mM), 10% FBS, 1% PS, and titrated to indicated target pH (large circles). Time courses show pH dynamics (measured from PhR ratio) evoked by placing media inside 5% CO₂ incubator, showing a tendency to acidify. b  Medium (DMEM D7777) supplemented with 22 mM NaHCO₃ plus non-volatile buffer (HEPES, PIPES or MES; 20 mM), and titrated to indicated pH before placement in 5% CO₂. Time courses show pH dynamics evoked by placing media in 5% CO₂ incubator, demonstrating pH instability. c Schematic of the chemical processes that underpin medium pH drifts. d pH-dependence of Caco2 cell growth (seeding density 4000 cells per well, growth area 0.32 cm² per well) measured from protein biomass (SRB assay) after 6 days of incubation in D7777 (25 mM glucose). Media were prepared by method A (20 mM HEPES/PIPES) or method B (20 mM HEPES/PIPES plus 22 mM NaHCO₃), and placed in 5% CO₂. To obtain a range of pH values, HEPES- and PIPES-buffered media were mixed in various ratios. Data are plotted as a function of assumed pH (titrated ‘at the bench’). Results compared against curve obtained with CO₂/HCO₃⁻ buffer, plotted against measured equilibrium pH (data from Fig. 2e). e Experiment performed on DLD1 cells, a more pH-sensitive line. Data are shown as mean ± SD (a, b) or mean ± SEM (d, e). All measurements were repeated three times (three technical replicates each). Statistical tests: two-sided t test (**P < 0.01)

Fig. 4

Enhancing buffering capacity of CO₂/HCO₃⁻-containing media with non-volatile buffers, with consideration of the CO₂-HCO₃⁻ equilibrium. a Medium (DMEMD 7777, 10% FBS, 1% PS, 25 mM glucose) supplemented with non-volatile buffer HEPES and MES (10 mM), and titrated to indicated target pH (large circles). NaHCO₃ then added to a concentration expected to be in equilibrium with 5% CO₂ at target pH (Fig. 1c). Time course of pH equilibration under 5% CO₂ from different starting levels. Repeated  three times (three technical replicates each). b Good agreement between target and measured equilibrium pH. c Time course of medium acidification in Caco2 cells (seeding density 4,000 cells per well, growth area of 0.32 cm² per well). Media buffered with 5% CO₂/22 mM HCO₃⁻, or the combination of CO₂/HCO₃⁻ plus 10 mM HEPES/MES (a 19% increase in time-averaged buffering, β). Medium lactate accumulation at the end point was greater with enhanced buffering. d Experiment performed on DLD1 cells. e Experiment performed on DLD1 cells with 30 mM HEPES/MES. f Experiment repeated from a more acidic starting pH, at which 30 mM HEPES/MES is expected to provide half of total buffering. Measurements repeated four times (three technical replicates each). Statistical tests: lactate measurements tested by two-sided t test (*P < 0.05, **P < 0.01); time courses tested by two-way analysis of variance (ANOVA) (P value for the effect of buffering is stated). g Increasing buffering capacity with non-volatile buffers also increases osmolarity due to the buffer molecules and the base required for titration. Calculated [NaOH] required to titrate MES, PIPES or HEPES buffer to a target pH. h Osmolality of three different media formulations. Arrows show gap in osmolality in HCO₃⁻-free media, which can be filled with buffer and acid–base required for titration, plus additional NaCl required to bring osmolality to a physiological level. Note, in the case of medium D7777 and D1152, a total of 88 and 132 mOsm kg⁻¹ can be added, respectively. i Free [Ca²⁺] measured by electrode, showing partial Ca²⁺ chelation by non-volatile buffers. Data are shown as mean ± SEM. Repeated three times

Fig. 5

Effects of buffering regime and medium pH on intracellular pH, measured using a high-throughput imaging method. a Monolayer of DLD1 cells imaged with Cytation 5 plate reader. Image on left shows superimposition of cSNARF1 and Hoechst-33342 fluorescence maps. Image on right shows pH in individual cells, identified by nuclear staining. b Calibration curve determined from nine colorectal cancer cell lines (LS174T, PMFKO14, LS513, HCT15, SW620, GP2D, HCT116, Caco2, RW2892; seeding density 100,000 cells per well, growth area 0.56 cm² per well). Three technical replicates each. Best-fit curve: pH = 6.978 + log((1.497 −ratio)/(ratio − 0.221)). c Histogram of intracellular pH in Caco2 monolayers bathed in D7777-based media (25 mM glucose) at pH 7.4, buffered by either 5% CO₂/22 mM HCO₃⁻, or 10 mM HEPES + MES titrated to 7.4 (CO₂ free). Note the substantial alkalinization in the absence of physiological buffer. Repeated three times (three technical repeats each). d Experiment performed on DLD1 monolayers. e Effect of medium pH on intracellular pH in Caco2 monolayers. The pH of CO₂/HCO₃⁻-buffered media was varied by changing [HCO₃⁻] (incubation in 5% CO₂). In contrast, the pH of HEPES/MES-buffered media were titrated to target pH with NaOH at the bench (incubation in 0% CO₂). Best fit: linear (CO₂/HCO₃⁻) or polynomial (HEPES/MES). Note that intracellular pH is more responsive to changes in extracellular pH in the absence of physiological (CO₂/HCO₃⁻) buffer. f Experiment repeated on DLD1 monolayers. Data are shown as mean ± SD. Repeated three times (three technical repeats each)

Fig. 6

Summary of buffering regimes in commercially-available media formulations, and flow chart showing instructions for preparing media at a target pH. a Venn diagrams summarizing the commercial availability of Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium (MEM) or RPMI-1640 buffers (supplied by Sigma-Aldrich and Thermo Fisher Scientific), grouped by buffering regime. Area is proportional to the number of media available in each category. Media with physiological HCO₃⁻ and no additional non-volatile buffer are highlighted with a thick black border. HEPES-buffered media are indicated with a red outline. b Flow chart guiding through the steps required to adjust the pH of culture media. NVB: non-volatile buffer (e.g. HEPES). Target [HCO₃⁻] for a given medium pH can be calculated from Eq. 3. Total osmolality can be approximated as 2 × [NaCl] + 2 × [NaHCO₃] + 2 × [KCl] + [Glucose]. See Supplementary Data 1 for further details of these steps