Role of carbon dioxide in animal cell culture

Carbon dioxide (CO²) and Bicarbonate

Carbon dioxide in the gas phase dissolves in the medium, establishes equilibrium with HCO₃⁻ ions, and lowers the pH. Because dissolved CO₂, HCO₃⁻, and pH are all interrelated, it is difficult to determine the major direct effect of CO₂.

The atmospheric CO₂ tension will regulate the concentration of dissolved CO₂ directly, as a function of temperature.

This regulation in turn produces H₂CO₃, which dissociates according to the reaction

(1)

H₂O+CO₂ ⇔ H₂CO₃ ⇔ H⁺ + HCO₃⁻

HCO₃⁻ has a fairly low dissociation constant with most of the available cat-ions so it tends to re-associate, leaving the medium acid. The net result of increasing atmospheric CO₂ is to depress the pH, so the effect of elevated CO₂tension is neutralized by increasing the bicarbonate concentration:

(2)

NaHCO₃ ⇔ Na⁺ + HCO₃⁻

The increased HCO₃⁻ concentration pushes equation (1) to the left until equilibrium is reached at pH 7.4. If another alkali (e.g., NaOH) is used instead, the net result is the same:

(3)

NaOH + H₂CO₃ ⇔ NaHCO₃ + H₂O ⇔ Na⁺ + HCO₃⁻ + H₂O

Intermediate values of CO₂ and HCO₃⁻ may be used, provided that the concentration of both is varied proportionately. Because many media are made up in acid solution and may incorporate a buffer, it is difficult to predict how much bicarbonate to use when other alkali may also end up as bicarbonate, as in equation (3). When preparing a new medium for the first time, add the specified amount of bicarbonate and then sufficient 1 N NaOH such that the medium equilibrates to the desired pH after incubation in a Petri dish at 37°C, in the correct CO₂ concentration, overnight. When dealing with a medium that is already at working strength, vary the amount of HCO₃⁻ to suit the gas phase, and leave the medium overnight to equilibrate at 37°C. Each medium has a recommended bicarbonate concentration and CO₂ tension for achieving the correct pH and osmolality, but minor variations will occur in different methods of preparation.

With the introduction of Good’s buffers (e.g., HEPES, Tricine) into tissue culture, there was some speculation that, as CO₂ was no longer necessary to stabilize the pH, it could be omitted. This proved to be untrue, at least for a large number of cell types, particularly at low cell concentrations. Although 20 mM HEPES can control pH within the physiological range, the absence of atmospheric CO₂ allows equation (1) to move to the left, eventually eliminating dissolved CO₂, and ultimately HCO₃⁻, from the medium. This chain of events appears to limit cell growth, although whether the cells require the dissolved CO₂ or the HCO₃⁻ (or both) is not clear. Recommended HCO₃⁻, CO₂, and HEPES concentrations are given in.

The inclusion of pyruvate in the medium enables cells to increase their endogenous production of CO₂, making them independent of exogenous CO₂, as well as HCO₃⁻. Leibovitz L15 medium contains a higher concentration of sodium pyruvate (550 mg/L) but lacks NaHCO₃ and does not require CO₂ in the gas phase. Buffering is achieved via the relatively high amino acid concentrations. Because it does not require CO₂, L15 is sometimes recommended for the transportation of tissue samples. Sodium β-glycerophosphate can also be used to buffer autoclavable media lacking CO₂ and HCO₃⁻, and Invitrogen markets a CO₂⁻ independent medium. If the elimination of CO₂ is important for cost saving, convenience, or other reasons, it might be worth considering one of these formulations, but only after appropriate testing.

In sum, cultures in open vessels need to be incubated in an atmosphere of CO₂, the concentration of which is in equilibrium with the sodium bicarbonate in the medium. Cells at moderately high concentrations (≥1×10⁵ cells/mL) and grown in sealed flasks need not have CO₂ added to the gas phase, provided that the bicarbonate concentration is kept low (∼4mM), particularly if the cells are high acid producers. At low cell concentrations, however (e.g., during cloning), and with some primary cultures, it is necessary to add CO₂ to the gas phase of sealed flasks. When venting is required, to allow either the equilibration of CO₂ or its escape in high acid producers, it is necessary to leave the cap slack or to use a CO₂⁻ permeable cap.

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