Folic Acid and Tetrahydrofolates in Cell Culture

Importance and uses of folic acid in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell, cultures

Folic Acid, a Serum-Free Medium Supplement, Useful In Biomanufacturing; Tissue Engineering and Specialty Media:

Folic acid and its reduced derivative(s), such as folinic acid, are an essential water soluble vitamins added to virtually all cell culture media.

Reduced and polyglutamated derivatives of folate are primary mediators of one-carbon unit metabolism within cells. Folic acid is formulated into Ames' Medium; Basal Medium Eagle (BME); BGJb Medium Fitton-Jackson Modification; Click's Medium; CMRL-1066 Medium: Dulbecco's Modified Eagle's Medium (DMEM); DMEM/Ham's Nutrient Mixture F-12 (50:50); F-12 Coon's Modification; Fischer's Medium; H-Y Medium (Hybri-Max^®); Iscove's Modified Dulbecco's Medium (IMDM); L-15 Medium; M2 Medium; M16 Medium; McCoy's 5A Modified Medium; MCDB Media 151, 153, and 302; Medium 199; Minimum Essential Medium Eagle (EMEM); NCTC Medium; Nutrient Mixtures, Ham's F-10); Nutrient Mixtures, Ham's F-12; Nutrient Mixture Ham's F-12 Kaighn's Modification (F12K); RPMI-1640; Serum-Free/Protein Free Hybridoma Medium; Waymouth Medium MB; Williams Medium E and various proprietary media.

Folinic acid is used in place of folic acid in MCDB Media 105, 110, 131 and 201.

Basal media used in the development of serum-free formulations for biomanufacturing of heterolgous proteins and other commercial applications typically contain folate as part of the basal formulation. Supplementation of media with folate for serum-free culture of eukaryotic cells is complicated by the fact that folate is marginally soluble at physiological pH and may be unstable. Hence the effective use of folate in cell culture requires an understanding of its chemistry. For a more complete discussion of folate as a cell culture additive, visit our Media Expert.

Primary Functions of Folic Acid in Cell Culture Systems:

Folates play a central role in one-carbon metabolism of cells. They exist as a large family of structurally related forms that transfer one-carbon groups among biomolecules that are important to cell growth, differentiation and survival. The biologically active folates are generally polyglutamylated and contain methyl, formyl, methylene, methenyl or formimino groups. These one-carbon units are inter-converted by folate metabolizing enzymes to adjust intracellular folate pool sizes and transfer one-carbon units to and from non-folate molecules. The biochemistry of folates is extremely complex involving a large number of enzymes that are regulated at the levels of gene expression and enzyme activity. Many of the inter-conversions of folates are oxidation-reduction reactions that utilize NADH, NADPH and ATP. The importance of folate metabolism to cells is illustrated by the observation that many cells can and do modify their phenotypes in response to factors that affect folate metabolism. The most well characterized phenotypic response of cells is the induction of dihydrofolate reductase gene expression by inhibitors such as methotrexate. Folates are involved in the synthesis of amino and nucleic acids and deliver one-carbon groups to S-adenosyl methionine (SAM) through the methionine homocysteine pathway.

Cells use several transport systems to take up folates. One route involves a reduced folate carrier system (RFC). Other folate receptor systems have been described.

Amino Acids:

Serine hydroxymethyl transferase (EC 2.1.2.1) catalyses the conversion of serine to glycine with the concurrent transfer of a one carbon unit to tetrahydrofolate. The resulting folate is N-5,10 methylene tetrahydrofolate (N-5,10 THF). This is a major entry point for carbon units into one-carbon metabolism. N-5,10 methylene THF can be used as a direct donor of one-carbon units or converted into the folates; N-5 methyl THF, N-5,10 methenyl THF or THF.

The most abundant form of folate in serum is N-5 methyl THF. This folate donates a methyl group to homocysteine during the formation of methionine. Methionine is a precursor of S-adenosyl methionine (SAM). SAM is a methyl and sulfate donor that is important in the synthesis of a wide range of molecules including creatine, polyamines, glutathione, and proteoglycans. It also supports the methylation of DNA. Methylation of DNA affects fundamental activities such as gene expression and repair. Folates are involved in the catabolism of histidine and glycine.

Nucleic Acids:

Thymidylate Synthetase (EC 2.1.1.45) converts deoxyuridine monophosphate, dUMP, into TMP required for DNA synthesis. The methyl group and reducing power for this reaction comes from N-5,10 methylene THF. Carbons 2 and 8 of the purine ring are derived form N-10 formyl THF and N-5,10 methenyl THF, respectively. Both of these folates can be synthesized from N-5, formyl THF in one enzymatic step.

Chemical Attributes of Folic Acid that make it a Useful Serum-Free Medium Supplement:

Folic Acid

• Formula C₁₉H₁₉N₇O₆
• Molecular Weight = 441.40
• Solubility approx. 3.6 mM at 25 °C.
• Unstable in visible and ultra violet light.

Folic acid is generally added to cell culture in its oxidized monoglutamate form. It is composed of a pterin ring; a para-amino benzoic acid residue (PABA)  and a glutamate residue. Active forms of folic acid are tetrahydrofolates that are reduced at atoms 5,6,7, and 8 of the pterin ring. One-carbon groups associated with active folates are bound to atoms N-5 and/or N-10 of the molecule. Folic acid is reduced in two steps to tetrahydrofolate by the enzyme dihydrofolic acid reductase (DHFR) (EC 1.5.1.3). Both of these reduction steps require NADPH. Additional glutamates are added to tetrahydrofolates inside the cell by folyl polyglutamate synthetase (EC 6.3.2.17). Polyglutamylated folates can contain up to nine glutamyl residues. More commonly they contain 3-7 glutamate residues. The polyglutamate forms of folate are found primarily inside the cell. The level of poly-glutamylation appears to control retention and distribution of folates.

Major one-carbon transport forms of folate include:

• N-5 formyl-THF, Folinic acid, Leucovorin, Citrovorum;
• N-5 methyl-THF;
• N-10 formyl-THF;
• N-5,10 methylene-THF;
• N-5,10 methenyl-THF;
• N-5 formimino-THF;

Folic acid is not very soluble at physiological pH. Solubility is reported to be about 3.6 µM (1.6 mg/L). A number of formulations have concentrations that exceed these levels. The concentrations may be substantially lower in the final media than is reported in the formula.

Folic Acid Products that Enhance the Growth of Hybridoma, Chinese Hamster Ovary (CHO) and other Mammalian Eukaryotic Cells in Serum-free Cultures.

Our Cell Culture Media Expert provides in depth discussion of this and other serum-free and protein-free media supplements. The Media Expert contains additional sections on raw materials, component use recommendations, formulation strategies and references. Whenever you have a questions about or problems with your eukaryotic mammalian cell culturing system visit the Media Expert for helpful guidance.