Vitamin B12, Cobalamin, in Cell Culture

Dennis R. Conrad, Ph.D.

biomediaexpert@earthlink.ne

Importance and uses of Vitamin B₁₂ in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell cultures

Vitamin B₁₂, a Serum-Free Media Supplement, Useful In Biomanufacturing, Tissue Engineering and Specialty Media

Cultured cells require pM levels of physiological Vitamin B₁₂. Vitamin B₁₂ supplementation concentrations range from 0 to 7.4 µM in non-proprietary commercially available classic media. Most classic media were developed with sera, especially FBS, as standard supplements. Sera contain Vitamin B₁₂. However, the amounts of Vitamin B₁₂ present in sera varies among species and with storage and handling. Variations of Vitamin B₁₂ present in sera partially explain the wide range of Vitamin B₁₂ found in classic media formulae. Chemical instability of Vitamin B₁₂ in cell culture media also contributes to the wide range of supplementation levels.

The following classic media contain no Vitamin B₁₂ in their basal formulations: Ames' Medium; Basal Medium Eagle (BME); Click's Medium; CMRL-1066 Medium: Dulbecco's Modified Eagle's Medium (DMEM); Fischer's Medium; Glascow Modified Eagle's Medium (GMEM);L-15 Medium; Medium 199; Minimum Essential Medium, Eagle (EMEM); and Swim's S-77 Medium.

NCTC Medium contains very high levels of Vitamin B₁₂, 7.4 µM.

RPMI-1640 and Iscove's Modified Dulbecco's Medium (IMDM) contain low levels of Vitamin B₁₂, compared to other supplemented media, at 3.7 and 9.6 nM, respectively. Alpha-MEM contains 100 nM vitamin B₁₂. IMDM and alpha-MEM are modifications of basal media that contain no vitamin B₁₂. Waymouth Medium MB and Williams Medium E both contain 148 nM vitamin B₁₂. H-Y Medium (Hybri-Max^®) and McCoy's 5A Modified Medium contain 923 nM and 1.48 µM of vitamin B₁₂, respectively. The high level of B₁₂present in H-Y Medium (Hybri-Max^®) media derives from its NCTC component.

DMEM/Ham's Nutrient Mixture F-12 (50:50) is a basal media frequently used as a base for development of proprietary serum-free or protein-free cell culture media used for biomanufacturing of heterologous proteins, especially with Chinese Hamster Ovary (CHO) cells. It contains 501 nM of Vitamin B₁₂ which derives from its F-12 component. Nutrient Mixture, Ham's F-10 and Nutrient Mixture, Ham's F-12 were developed for clonal growth of CHO cells without use of FBS. These media and their derivatives: F-12 Coon's Modification; Nutrient Mixture Ham's F-12, Kaighn's Modification (F12K) and Serum-Free/Protein Free Hybridoma Medium all contain 1 µM Vitamin B₁₂.

The level of Vitamin B₁₂ in media of the MCDB series was adjusted to one of two concentrations: media 105, 110, 131, 201 and 302 contain 100 nM and media 151 and 153 contain 300 nM of vitamin B₁₂.

The relatively high level of vitamin B₁₂ in serum-free hybridoma media; Hybri-Max^® and Serum-Free/Protein Free Hybridoma Medium suggests that serum-free systems for monoclonal antibody production require significant supplementation of media with vitamin B₁₂. Cell's in culture require physiological Vitamin B₁₂ in the pM concentration range. The presence of vitamin B₁₂ in basal media at nm and µM levels indicates that its delivery via cell culture media is complex. The biggest issue for serum-free formulations may relate to the chemical instability of Vitamin B_12. For a more complete discussion of Vitamin B₁₂ as a cell culture media supplement go to the Media Expert.

Primary Functions of Vitamin B₁₂ in Cell Culture Media

Vitamin B₁₂ is the generic name for a family of cobalamin molecules required for growth, genetic stability and survival of cells in vitro. Members of this family are interchangeable and have different axial ligands and cobalt oxidation levels. As unique coenzymes of methionine synthase (EC 2.1.1.13) and methylmalonyl-CoA mutase (EC 5.4.99.2), the Vitamin B₁₂ cobalamins, methylcobalamin and 5’-adenosylcobalamin, support one-carbon metabolism and the degradation of amino and odd-chain fatty acids, respectively. Vitamin B₁₂ deficiency in vitro may contribute to acidosis, genome instability, and mitochondria-mediated apoptosis.

One-Carbon Metabolism:

Methionine synthase (MS) is a regulated enzyme that links a cell’s methionine:S-adenosylmethionine-mediated (SAM) methylation pathway with its folate-mediated one-carbon transfer pathway. It is a methylcobalamin containing cytoplasmic enzyme that methylates homocysteine to methionine and converts N5-methyltetrahydrofolate to tetrahydrofolate. MS keeps methyl groups from accumulating as N5-methyl tetrahydrofolate, the methyl-folate trap, by regenerating tetrahydrofolates (THF). Failure to regenerate THF can lead to disruption of nucleic acid synthesis. N5-methyl THF is the predominant folate present in plasma. MS controls the flow of folate-transported methyl groups into the cell’s primary methylation pathway, the methionine:S-adenosylmethionine (SAM) pathway.

S-Adenosyl Methionine (SAM):

S-Adenosyl methionine (SAM) is important for cell function and survival. It is involved in several important metabolic processes including transmethylation, transulfuration and polyamine synthesis.

SAM donates methyl groups to a variety of molecules including small molecules, proteins, phospholipids and nucleic acids. A large number of unique SAM:methyltransferase have been identified and characterized. Their transfer of SAM methyl groups into DNA and RNA bases is important for the regulation of gene expression, DNA repair and genome stability.

SAM supports the formation of creatine, a precursor of creatine phosphate, from quanidinoacetate.

Polyamines are essential for multiple cell functions including cell proliferation and differentiation. Cell growth and survival is sensitive to the size and makeup of intracellular polyamine pools. When the concentration of polyamines becomes too low, cells become growth arrested. When the concentration of polyamines becomes too high, cells may enter apoptosis. Polyamine biosynthesis is closely regulated by the enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAM-DC). ODC controls the formation of putrescine and SAM-DC controls the formation of spermidine and spermine. The synthesis of spermidine and spermine from putrescine requires the donation of aminopropyl groups from decarboxylated S-adenosylmethionine which is formed from SAM by S-adenosylmethionine decarboxylase (SAMe-DC) (EC 4.1.1.50).

SAM can also be catabolized through multiple steps to L-cysteine which is an important precursor of the antioxidant molecules glutathione, taurine and hypotaurine. While methionine is a direct precursor of SAM, the addition of methionine directly to cells is not adequate, because if the methionine synthase enzyme is not active methionine may cause a buildup of homocysteine and S-adenosylhomocysteine which can be toxic to cells.

Apoptosis: Methionine: SAM Cycle:

The transfer of methyl groups from SAM to cell substrates by methyl-transferase produces S-adenosylhomocysteine (SAH). SAH is converted to homocysteine (HCys) which is then methylated with methyl groups derived from the folate pool to methionine by methionine synthase. Methionine is adenosylated to SAM. This cycle pumps methyl groups from the folate methyl pool into the cell’s methylation pathways which support gene expression and DNA repair. When MS is not active, both homocysteine and S-adenosylhomocysteine can accumulate and induce apoptosis. If L-homocysteine accumulates in cells with dysfunctional methionine synthase, it can reverse the adenosylhomocysteinase (EC 3.3.1.1) reaction and form SAH, an inhibitor of DNA methylation. Adenosine will drive this reaction towards SAH. Adenosine and cAMP bind adenosylhomocysteinase. Hypomethylation of DNA occurs as the ratio of SAH/SAM increases, and this can lead to DNA instability. The enzyme poly-ADP-ribose polymerase (PARP) is an important enzyme for maintaining genome integrity. It is a DNA nick sensor that uses NAD(+) to polyribosylate proteins involved in DNA repair. This enzyme is found in both the nucleus and mitochondria. ATP and NAD are critical to maintain mitochondrial membrane potential. Under conditions of oxidative stress or DNA damage, PARP can deplete the mitochondria of ATP and NAD+ resulting in membrane destabilization and mitochondria-mediated apoptosis. The addition of adenosine or cAMP to media may contribute to apoptosis when methionine synthase activity is low as a consequence of cobalamin deficiency. The level and effect of adenosine on the formation of SAH will also depend upon the activity of adenosine kinase which removes adenosine as a substrate.

Proprionyl-CoA Metabolism:

Proprionyl-CoA is formed in the mitochondria by β-oxidation of odd-chain fatty acids and catabolism of the amino acids; isoleucine, valine, threonine, homocysteine, and methionine. Proprionyl-CoA is catabolized to succinyl-CoA by three enzymes; proprionyl-CoA carboxylase (EC 6.4.1.3), (S)-methylmalonyl-CoA isomerase and (R)-methylmalonyl-CoA mutase (EC 5.4.99.2). MM-CoA-M completes the formation of succinyl-CoA from proprionyl-CoA. The deacylation of succinyl-CoA to succinate is accompanied by the formation of GTP. In animal cells, much of this GTP is converted to ATP by nucleoside diphosphokinase. This process is called substrate phosphorylation. It supports the formation of ATP without the involvement of oxygen. Cells that lack Vitamin B₁₂ cannot recover energy as succinate and ATP from odd-chain fatty acids and proprionyl-CoA forming amino acids. If MM-CoA-M is inactive methylmalonyl-CoA may accumulate and cause acidosis.

Cobalamin Delivery and Metabolism:

Cobalamin Transport:

Vitamin B₁₂ is the general name for a family of cobalamins (Cbl) that contain different coordinated ligands. Nutritional forms of Vitamin B₁₂ are the Cbl(III)alamin axial-ligated molecules cyano-Cbl, aquo-Cbl and hydroxo-Cbl, see chemistry. These forms of cobalamin are transported into cells as complexes with transcobalamin-II and internalized by specific transcobalamin-II receptors, TC-IIR. Many cells synthesize and secrete apoTC-II into the media and TC-IIR activity generally increases as cells transition into active division. Inside the cell, the Cbl(III)alamin:TC-II complex is transported to lysosomes where ligand-containing:Cbl(III)alamin is released from TC-II. Subsequently, Cbl(III)alamin undergoes axial-ligand exchange and reduction to Cbl(II)alamin by an NADH- or NADPH-linked Cbl(III)alamin reductases such as cyanocob(III)alamin, hydroxocob(III)alamin and aquacob(III)alamin reductases (EC 1.6.99.8, NADH-linked; EC 1.16.1.3, NADH-linked or EC 1.16.1.5, NADPH-linked)). The cytoplasmic enzyme glutathionyl ligand transferase has been shown to convert CN-, aquo- and hydroxocobalamins to glutathionylcobalamin. Glutathionylcobalamin may be a preferred precursor for the formation of the coenzymes, methylcobalamin and 5’adenosylcobalamin. Glutathionylcobalamin is a relatively stable form of cobalamin formed inside the cell with high affinity for cobalamin reductases which convert it to cob(II)alamin. Cbl(II)alamin either binds to methionine synthase in the cytoplasm where it is activated by further reduction and methylation to methylcobalamin or it enters the mitochondria where it is reduced to Cbl(I)alamin, the substrate for cob(I)alamin adenosyltransferase (EC 2.5.1.17). This enzyme forms 5’adenosylcobalamin from ATP and cob(I)alamin

In Vitro Culture:

Increased demand for methionine by transformed and stimulated cells requires both external supplementation and internal synthesis. Dividing lymphocytes and other mononuclear cells have an increased dependence on cobalamin containing methionine synthase. Actively dividing cells are dependent upon the activity of methionine synthase to grow and maintain genome integrity. The effective concentration of cobalamin is dependent on its form of delivery. The most effective form of delivery is cobalamin bound to transcobalamin II.

Chemical Attributes of Vitamin B₁₂ that make it a Useful Serum-Free Media Supplement

Cobalamins are water soluble molecules that do not cross cell membranes efficiently. They generally enter cells as a complex with transcobalamin II or in the case of enterocytes in the gut, with intrinsic factor.

Cobalamin is composed of two parts: a nucleotide, 5,6 dimethylbenzimidazole and a cobalt-coordinated highly substituted and reduced corrin ring. Corrin-cobalt forms up to six coordination bonds with octahedral symmetry. Four of these bonds are in a plane and two are in axial orientations. The planar coordination bonds of cobalt chelate four nitrogens of the corrin ring. One axial coordination bond chelates an imidazole-nitrogen of the nucleotide and the other axial bond can be free or occupied by various ligands. When the axial bonding position is unfilled, cobalamins are generally in the cob(I)alamin or cob(II)alamin oxidation states. When this bond contains a ligand, the level of oxidation of cobalt affects the reactivity of the molecule. The oxidation level of the nutritional cobalamins and the coenzyme cobalamins is generally cob(III)alamin.

Cobalamins are a family of molecules with different ligands at the exchangeable axial coordination bond of cobalt and with different levels of cobalt oxidation. Vitamin B₁₂ is a general term that refers to the nutrient forms of cobalamin. Cobalt in nutrient cobalamins is usually oxidized to Co(III)alamin and its axial bond is ligated to cyanide, water or a hydroxyl group. Cyanocobalamin is produced during commercial manufacturing. In vivo, the formation of cyanocobalamin may actually be a mechanism for defense against cyanide poisoning. Inside the cell, these ligands are replaced by glutathione or removed with the reduction of cobalt to Co(II)alamin and Co(I)alamin. Co(II)alamin and Co(I)alamin forms of cobalamin are the precursors of the coenzymes methylcob(III)alamin and 5’adenosyl(III)cobalamin, respectively.

Cobalamin Stability:

Serum Vitamin B₁₂ is highly unstable and that it should be protected from light and kept frozen. Cyanocobalamin is often used to supplement media. It has a maximal stability at pH = 7, but is photolysed to hydroxocobalamin with a zero-order rate constant. Hydroxocobalamin may be converted to sulfitocobalamin in the presence of sulfur dioxide or other sulfites. Hydroxocobalamin can convert to ammoniacobalamin if ammonia is present. In aqueous oxygenated systems, Cbl(III)alamin rapidly and spontaneously forms the stable complex superoxocobalamin, Cbl(III)-O₂^-. Superoxocobalamin reacts with nitric oxide, NO, with the possible formation of peroxynitrite, OONO^-, a precursor of free radicals and nitrous oxide. Low μM concentrations of NO-Cbl can induce apoptosis, especially in rapidly dividing cells. Nitrous oxide (N₂0) can inhibit the cobalamin enzyme methionine synthase. Nitrite, NO₂, binds to Cbl(III) and Cbl(II).

The in vitro chemistry of cobalamin is very complex and the potential is high for cobalamin to become inactive and/or participate in chemical reactions. It can participate in a wide range of radical redox reaction with oxygen, nitrogen and sulfur molecules and many of these reactions can generate toxic species.

Vitamin B₁₂ Products that Enhance the Growth of Hybridoma, Chinese Hamster Ovary (CHO) and other Mammalian Eukaryotic Cells in Serum-free Cultures.