CO₂ concentration and pH control in the cell culture laboratory

Most cell culture laboratories will have a humidified Carbon Dioxide (CO₂) incubator set to 5% CO₂, however it is not always obvious why our cultures need this amount of CO₂ nor what the acceptable tolerances are for the ranges in CO₂ concentration.  You may also have heard or read that Dulbecco’s Modified Eagle’s Medium (DMEM) requires a higher (10%) concentration of CO₂, yet most of us use 5% CO₂ with this medium and there is scant information available to advise the impact of using lower CO₂ concentrations with DMEM.

CO₂ is not a metabolic requirement for cell cultures, its purpose is to dissolve into cell culture medium where a small proportion of it reacts with water to form carbonic acid which in turn interacts with its conjugate base (the dissolved bicarbonate ions in the medium) to control a stable physiological pH through the bicarbonate buffering system. The amount of sodium bicarbonate (NaHCO₃) in the medium dictates the amount of CO₂ that should be used to maintain the pH.  Physiological pH is generally considered to be in the range of 7.2 to 7.4 for normal tissues, but some disease states (such as cancer) may require culture at a lower pH. Eagle’s Minimal Essential Medium (EMEM) supplemented with Earle’s Balanced Salt Solution (EBSS) and most other cell culture media are formulated with 26mM sodium bicarbonate, DMEM on the other hand has a far higher concentration of 44mM sodium bicarbonate.

The bicarbonate buffering system is not the most efficient chemical system for controlling pH.  There are other synthetic buffers such as HEPES which work over a wider range of laboratory conditions. The bicarbonate system, however, occurs naturally in the human body and is therefore used in in vitro cell culture to mimic human and mammalian physiology, minimising toxic side effects.

Generally, cell culture media are also supplemented with the indicator dye Phenol Red.  A yellow hue to the medium indicates the pH is too low (acidic), a purple hue shows the pH is too high (alkaline) whereas an orangey-red hue indicates the medium is of the correct physiological pH (Figure 1).

Figure 1. Cell culture media are generally supplemented with Phenol Red to act as a pH indicator. The colour of the medium helps advise if cultures are at physiological pH and can assist with troubleshooting.

Bicarbonate buffering works through Le Chatelier’s principle. Increased acidity in the medium is manifested by an increase in Hydrogen (H⁺) ions; free bicarbonate ions then react with the extra H⁺ ions to form carbonic acid “shifting the reaction to the left”, stabilising pH. (See Figure 2). Similarly, a decrease in H⁺ ions will result in a “shift to the right”.

Figure 2. The principle of bicarbonate buffering

Using Dalton’s law of partial pressures to determine the partial pressure of CO₂ in the incubator atmosphere, Henry’s law together with the Henry’s constant of the medium to determine the amount of CO₂ that dissolves into the medium at 37°C and the Acid Equilibrium Constant of the bicarbonate buffering system it is possible to estimate the theoretical pH values that should be observed in different medium formulations at different percentages of CO₂ and therefore evaluate the ranges of CO₂ concentration that can be used for the media to maintain an optimal physiological pH range.

These calculations (see Appendix) can then be used to estimate the pH range seen in different culture media with different percentages of CO₂ in the incubator (Figure 3).

Figure 3. Theoretical pH ranges for different culture media in increasing CO₂ levels in a 37°C humidified incubator. The lower and upper physiological range of pH is denoted by the red and green horizontal lines (pH 7.2-7.4). DMEM (44mM NaHCO₃, orange line) is held at physiological pH at CO₂ concentrations between 7.5% and 11%. Using DMEM in a 5% CO₂ environment will result in a pH of 7.5 which although acceptable for most cell cultures is slightly outside the desired range of physiological pH.  EMEM + Earle’s Balanced Salt Solution (BSS) (26mM NaHCO₃, dark green line) is held at physiological pH with CO₂ levels between 4.5% and 6.5%, whereas a much lower percentage of CO₂ (near atmospheric levels) is required for EMEM + Hank’s BSS. (4mM NaHCO₃, blue line).

These theoretical data show that for most cell culture media with 26mM NaHCO₃, at 37°C the percentage of CO₂ in the incubator should be maintained at a nominal 5% (between 4.5% and 6.5%).  In theory DMEM (44mM NaHCO₃) a higher percentage of CO₂ should be used (between 7.5% and 11.5%, at a nominal level of 10%).  However, it has become convention to use 5% CO₂with DMEM and most cell line suppliers will advise this.  Cell culturists should be aware that using 5% CO₂ with DMEM will result in a pH of 7.5-7.6 which is slightly above the physiological range, however, in most cases the lactic acid and CO₂ evolved by healthy, growing cultures will offset and correct this high pH.  If you are working with very low density cultures in DMEM or have particularly slow growing cultures, then increasing the CO₂ level of the incubator may be a worthwhile exercise. Bear in mind, however, that higher percentage of CO₂ will displace oxygen in the incubator, reducing the available oxygen to the cells, so percentages of CO₂ above 10% should not be used.

Finally, it is vitally important that CO₂ incubators are properly serviced and calibrated to ensure that both temperature and CO₂ displays are accurate.  At ECACC we use an independent CO₂monitor calibrated to United Kingdom Accredited Service (UKAS) Standards to regularly check and re-calibrate the internal incubator CO₂ probe and display (Figure 4).

Figure 4. The Geotech G100 CO₂ monitor. An example of a calibrated CO₂ monitor that can be used to check and verify CO₂ incubator internal probes and displays.

 

References

1.  Freshney, R. I. (2010) Culture of Animal Cells John Wiley & Sons, Inc.
2.  Johnson, Leonard R., ed. (2003). Essential Medical Physiology: Elsevier Academic Press. 

 

Appendix: Calculations and Formulas Used Note:

• These calculations are based on Henry’s Law at 37⁰C for blood and assumes the Henry’s constant of complete culture medium to be similar.
• Only a very small proportion of the dissolved CO₂ reacts to form carbonic acid, so Henry’s Law “holds true”.
• The system must run at atmospheric pressure.
• The calculations are based on cell culture medium alone. The effect of CO₂ and lactic acid produced by cell cultures is not cot considered.

 

1. To determine the amount of dissolved CO₂; use Henry’s Law

Where K_CO2 is the Henry’s Constant of CO₂ (3.0x 10^-2 mole/L x Atm at 37⁰C) (blood/culture medium)and P_CO2 is the partial pressure of CO₂ in the incubator. Calculate the partial pressure of CO₂ in Atmospheres (Atm) units:

Then calculate the concentration of aqueous CO₂  ([CO2 (aq)]) using Henry’s Law, and convert the concentration to Moles/L.

2. Now we have the [CO2 (aq)], using the acid equilibrium constant of the Carbonic Acid / Bicarbonate reaction we can estimate the hydrogen ion (H+) concentration.

3. The pH can then be calculated:

Jim Cooper