Sodium bicarbonate and buffering Cells produce and require small amounts of carbon dioxide for growth and survival.6 In culture media, dissolved CO2 is in equilibrium with bicarbonate ions and many medium formulations take advantage of this CO2/bicarbonate reaction to buffer the pH of the medium. CO2 dissolves freely into the medium and reacts with water to form carbonic acid. As the cells metabolize and produce more CO2, the pH of the medium decreases as the chemical reaction below is driven to the right:
H2O + CO2 <—> H2CO3 <—> H+ + HCO3-
The optimal pH range of 7.2 to 7.4 can be maintained by supplementing the medium with sodium bicarbonate and regulating the level of CO₂ in the atmosphere above the medium as shown by the reaction below:
H2O + CO2 + NaHCO3 <—> H+ + Na+ + 2HCO3-
In tissue culture, cells are grown either in open systems (where there is free exchange of the atmosphere immediately above the medium with the atmosphere of the incubator) or in closed systems (where the two atmospheres are kept separate). The buffering system employed in the medium needs to be matched to the culture system. Otherwise the cells may be subject to metabolic stress which will impair their performance.
In closed systems the level of CO2 is regulated by the metabolism of the cells. The culture vessel must be sealed (flasks tightly capped) to retain any CO2 generated by the cells. Consequently, closed systems provide additional protection against contamination and have simpler incubator requirements than open systems. Closed systems usually require media with buffers based on Hanks’ balanced salt solution having relatively low levels of sodium bicarbonate.
In open systems, humidity (to reduce evaporation) and a means of regulating CO2 levels (if the culture medium contains sodium bicarbonate) are required during incubation to maintain the pH of the culture medium. Open systems usually require the higher levels of sodium bicarbonate found in Earle’s salt solution combined with a 5 to 10% CO2 atmosphere supplied by the incubator. In general, 1.2 g/L to 2.2 g/L of sodium bicarbonate is used with 5% CO2 whereas 3.7 g/L sodium bicarbonate is used with 10% CO2. The exact amount will depend upon the medium formulation.
In some cases, researchers “gas” the atmosphere of the culture vessel with a stream of sterile-filtered 5% CO2/95% air mixture and then tightly seal the flask prior to incubation in a nonhumidified and non-CO2 incubator.7 While these culture vessels work with simpler non-humidified, non-CO2 incubators, the medium requirements are those of an open system.
All ATCC media, with the exception of Leibovitz’s L-15 (ATCC® 30-2008™), are designed to be used with 5% CO2 levels. Most have a sodium bicarbonate concentration of 1.5 g/L and are supplemented with extra sodium pyruvate. ATCC modification of McCoy’s 5A (ATCC® 30-2007™) has a slightly higher levels of sodium bicarbonate (2.2 g/L) and does not contain sodium pyruvate.
While most commercial formulations of liquid media do contain the appropriate amount of sodium bicarbonate, it is generally omitted from the powdered form and needs to be added before use. Some medium formulations incorporate other buffering systems such as phosphate or HEPES in addition to CO2/ sodium bicarbonate. These media have the advantage of maintaining optimal pH in an open system when the culture vessel is removed from the enriched CO2 atmosphere of the incubator.
HEPES and other organic buffers can be used with many cell lines to effectively buffer the pH of the medium.8 Indeed, some standard medium formulations include HEPES. However, this compound can be toxic, especially for some differentiated cell types, so evaluate its effects before use.9 HEPES has been shown to greatly increase the sensitivity of media to the phototoxic effects induced by exposure to fluorescent light.10,11
Phenol red is used to monitor the pH of media. During cell growth, the medium changes color as it changes pH due to metabolites released by the cells. At low pH levels, phenol red turns the medium yellow, while at higher pH levels it turns the medium purple. For most tissue culture work (pH 7.4), the medium should be bright red.
Unfortunately, phenol red can mimic the action of some steroid hormones, particularly estrogen. For studies with estrogen-sensitive cells, such as mammary tissue, use media without phenol red. Additionally, the sodium-potassium ion homeostasis is upset when phenol red is included in some serum-free formulations; this effect is neutralized by the inclusion of serum or bovine pituitary hormone in the medium.12 Phenol red is frequently omitted from studies with flow cytometry as its color interferes with detection.
L-Glutamine (ATCC® 30-2214™) is an essential amino acid required by virtually all mammalian and insect cells grown in culture. It is used for protein production, as an energy source, and in nucleic acid metabolism. It is also more labile in liquid cell culture media than other amino acids. The rate and extent of L-glutamine degradation are related to storage temperatures, age of the product, and pH.
Because L-glutamine is so labile, it is often omitted from commercial liquid medium preparations to lengthen the product shelf life. In these cases, it must be aseptically added prior to use. L-Glutamine is not as labile in dry form and most powdered medium formulations do include it.
In some cases, the addition of L-glutamine to complete cell culture medium can extend the usable life of the medium. If L-glutamine is suspected to be a limiting factor during cell culture, a simple test of ‘spiking’ the medium with a small amount of L-glutamine will determine whether or not more is required. Simply add a small amount of L-glutamine (~2 mM final concentration) to the culture medium. If the cell growth rate increases, L-glutamine is most likely deficient and more should be added. Alternately, the concentration of L-glutamine can be measured directly by standard analytical means such as HPLC (High Performance Liquid Chromatography).
L-Glutamine concentrations for mammalian cell culture media can vary from 0.68 mM in Medium 199 to 4 mM in Dulbecco’s Modified Eagle’s Medium. Invertebrate cell culture media, such as Schneider’s Drosophila medium, may contain as much as 12.3 mM L-glutamine.
Use caution when adding more L-glutamine than is called for in the original medium formulation. L-Glutamine degradation results in the build-up of ammonia which can have a deleterious effect on some cell lines. For most cell lines, ammonia toxicity is more critical for cell viability than L-glutamine limitation.
Nonessential amino acids
All medium formulations contain the ten essential amino acids as well as cysteine, glutamine, and tyrosine. The inclusion of the other non-essential amino acids (alanine, asparagine, aspartic acid, glycine, glutamic acid, proline, and serine) in some media formulations reduces the metabolic burden on the cells allowing for an increase in cellular proliferation.
Pyruvate is an intermediary organic acid metabolite in glycolysis and the first component of the Embden-Meyerhof pathway. It can pass readily into or out of the cell. Its addition to tissue culture medium provides both an energy source and a carbon skeleton for anabolic processes. Pyruvate may help in maintaining certain specialized cells, in clonal selection, in reducing the serum concentration of the medium,7 and in reducing fluorescent light-induced phototoxicity.10 Cellular metabolism of pyruvate produces carbon dioxide which is given off into the atmosphere and becomes bicarbonate in the medium. Sodium pyruvate is added to give a final concentration of 1 mM in most media, but is increased to 5 mM in Leibovitz’s L-15 medium primarily to facilitate use in CO2-free environments.