Nutrients

Glucose

The primary carbohydrate normally included in mammalian culture media is glucose, routinely supplemented from 5 to 25 mM. The majority of glucose is metabolized via glycolysis to form pyruvate, which is then reduced to lactate. This eventually leads to a buildup of lactic acid in the medium. Studies of metabolic pathways have revealed that only a small portion of glucose (~20-30%) enters the other pathways including the tricarboxylic acid cycle and the pentose phosphate shunt (39).

In addition to glucose evidence has shown that mammalian cells also utilize glutamine as their major energy source. Glutamine is often included at high concentrations relative to the other amino acids (2-4 mM), and has been shown to be a high requirement of many cell lines. In most cultures, glutamine and glucose are utilized particularly rapidly and can cause cell growth limitations even before their complete exhaustion. This trend is more pronounced in serum-free or low-protein media, and higher utilization rates have been observed (39).

Amino Acids

The nutrient requirement of cultured cells includes the essential amino acids (i.e., those not normally synthesized in mammals in vivo) along with others depending on the specific requirements of the individual cell line. The nonessential amino acids are usually included because some cell lines may not be able to produce their own, which would lead to a limitation in their growth potential. The effect of an amino acid limitation in the medium will be a reduction in the growth rate and/or the maximum cell density. Other nonessential amino acids can be produced by the cell but not in sufficient quantities to maintain maximal growth. Some amino acids are not stable in the medium (e.g., glutamine), thus requiring supplementation to maintain a suitable level.

Amino acids are often added as defined components into the medium. There are, however, undefined sources of amino acids, such as serum, tryptose phosphate broth or lactalbumin, or various plant and animal cell hydrolysates.

Glutamine is routinely used in culture media as it has been shown to be a major energy source for mammalian cells. However, glutamine can lead to a buildup of ammonia in the medium, which can result in growth inhibition of some ammonia-sensitive cell lines (40). In this case glutamate, or glutamine dipeptides (ala-gln and gly-gln), have been shown to be successful substitutes and will lower the accumulation of ammonia in the medium.

Branched amino acids are consumed particularly rapidly by a number of cell lines including Madin-Darby canine kidney (MDCK) cells (41), human fibroblasts (42), mouse myeloma cells (43), and BHK cells (44). It has been found that branched amino acids are more extensively oxidized by hybridoma cells when the specific glutamine utilization rate is low (45). This group also observed that the serine consumption was higher when the glutamine consumption was reduced.

Some cell lines can be more sensitive to amino acid limitations than others and can be important for feeding strategies in larger-scale systems. For example, under low glutamine concentrations, BHK cells will increase their consumption of other amino acids, especially the essential amino acids leucine, isoleucine, and valine and the nonessential amino acids serine and glutamate (46).

Salts

The media salt concentration is isotonic to prevent osmotic imbalances. The osmolality of standard growth media is approximately 300m0sm/kg and is optimal for most cell lines. Care should be taken when supplementing a medium with extra salts as this will change the osmolality. However, many cell lines have been shown to tolerate variation of approximately 10% of this optimal value.

The salts that are primarily used are those of Na+, K+, Mg2+, Ca2+, Cl", S0|", PO43", and HC03". These are present in most basal media formulations.

Trace Elements

Other inorganic elements that are present in serum in trace amounts may also be included in media formulations. These include Mn, Cu, Zn, Mo, Va, Se, Fe, Ca, Mg, Si, Ni. The less common elements that are not seen as essential have also been supplemented into media, including Al, Ag, Ba, Br, Cd, Co, Cr, F, Ge, J, Rb, and Zr. Many of these elements play an important role in enzyme activity and are essential to the survival of most cells.

The most commonly added element to serum-free formulations is selenium. The major functions of selenium can be attributed to its antioxidative properties and its role in cell growth. Selenium functions with mammalian cells in the form of selenoproteins, which have a number of physiological roles, of which 11 have been identified. Most of these proteins have been proven or implicated in antioxidant activities (47), such as glutathione peroxidase and thioredoxin reductase (48).

In some cases the supplementation of certain trace elements can decrease the requirement of certain growth factors in the medium. A ferrous salt, for example, can be used to replace transferrin as an iron carrier in many cell lines, such as B-lym-phocytes, MDCK, and human diploid fibroblasts (49-51).

Calcium has been shown to act as an important element involved in cell proliferation. It is important to many processes, including signal transduction, cell division, and cell adhesion. Calmodulin, a calcium-activated protein, regulates many serine/threonine kinases involved in cell division, in a calcium concentration dependent manner (52). Elevated calcium levels has been shown to be beneficial to some cell lines. These include keratinocytes (53,54), and human diploid fibroblasts (55). The reduction of calcium in culture media can be effective in reducing the degree of cell-cell adhesion. Cell clumping is a major problem for some production systems as this may reduce productivity and cell viability (56). Reducing the calcium in the medium can avoid this problem and is routinely done in many agitated-culture systems.

Vitamins

The vitamin and hormone components are present at relatively low concentrations and are utilized as cofactors, the requirements for which show considerable variations between cell lines. Thus, the content of these cofactors varies considerably among different media formulations. The requirement for extra vitamins is increased whenever the serum concentration is reduced. Cells that are limited in vitamins will vary in cell growth and survival but not in maximum cell density.

The vitamins included in each basal media formulation will vary depending on the cell line for which it was designed. The more basic media formulations, such as BME, designed for HeLa cells and mouse fibroblasts, contain biotin, folic acid, nicotinamide, pantothenate, pyridoxal, riboflavin, and thiamine. These are typically supplemented with serum, which provides the other cofactor and vitamin requirements. The more complex formulations, such as F12, DMEM, or M199, are designed for serum-free formulations and contain a greater source of vitamins. M199, for example, contains all of the vitamins in BME but also includes vitamins A, B6, C, D3, E, K, and ^-aminobenzoic acid (PABA). Each of these vitamin formulations was designed empirically for a specific cell line. Therefore, the requirements of another cell line may be different and will have to be determined independently.

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