• Thiamine, Useful In Biomanufacturing; Tissue Engineering and Specialty Media:
• Primary Functions of Thiamine in Cell Culture Systems:
• Chemical Attributes of Thiamine that make it a Useful Serum-Free Medium Supplement:
• Thiamine Products that Enhance the Growth of Hybridoma, Chinese Hamster Ovary (CHO) and other Mammalian Eucaryotic Cells in Serum-free Cultures.

Thiamine, a Serum-Free Medium Supplement, Useful In Biomanufacturing; Tissue Engineering and Specialty Media:
Thiamine is a water soluble, but chemically unstable essential vitamin used in cell culture. It is typically added to basal media formulations, but may also be introduced as a component of serum.

The concentration of thiamine in basal formulations ranges over three logs from 0.027 µM in Medium 199 and its derivative CMRL-1066 Medium to 26.8 µM in Waymouth Medium MB. The concentration of thiamine is also low in NCTC Medium; Ames' Medium and McCoy's 5A Modified Medium at 0.067, 0.27 and 0.54 µM, respectively.

Nutrient Mixture, Ham's F-12 and its derivatives; F-12 Coon's Modification; Nutrient Mixture Ham's F-12 Kaighn's Modification (F12K); MCDB Media (105, 110, 131, 151, 153, 201 and 302); and Serum-Free/Protein Free Hybridoma Medium contain 0.91 µM of thiamine. Earle's Basal Medium, Eagle (BME); Minimum Essential Medium, Eagle (EMEM); Fischer's Medium; Nutrient Mixture, Ham's F-10; RPMI-1640; and Williams Medium E all contain 2.68 µM of thiamine. Swim's S-77 Medium contains 1.7 µM of thiamine.

Media with relatively high levels of thiamine include: Click's Medium and Glascow Modified Eagle's Medium (GMEM) at 5.36 µM; Dulbecco's Modified Eagle's Medium (DMEM)/Ham's Nutrient Mixture F-12 (50:50) at 5.82 µM; H-Y Medium (Hybri-Max^®) at 9.46 µM; and BGJb Medium Fitton-Jackson Modification; Dulbecco's Modified Eagle's Medium (DMEM); and Iscove's Modified Dulbecco's Medium (IMDM) which are all at 10.72 µM.

While concentrations of thiamine in the lower range may not be adequate for growth of some cell types, the high concentrations present in DMEM and IMDM may become toxic under certain storage conditions. Hence, the use of thiamine should be a balance of optimal concentration required for growth and limits imposed by storage and stability requirements. For a more complete discussion of thiamine as a cell culture component go to Sigma's Media Expert.

Primary Functions of Thiamine in Cell Culture Systems:
Thiamine is a required nutrient that is taken up by cells via a carrier-mediated system that may be regulated by calmodulin. The active vitamin form, thiamine pyrophosphate (TPP), is synthesized from ATP and thiamine by thiamine diphosphokinase (EC 2.7.6.2). TPP is an enzyme cofactor. TPP containing enzymes are involved in energy metabolism and amino acid synthesis. Pyruvate cannot be metabolized effectively if thiamine is not provided to cells in vitro. The metabolism of pyruvate to acetyl-CoA is a critical reaction in cells that is mediated by the pyruvate dehydrogenase complex. Thiamine is one of three vitamins required for this complex to function.

Energy Production: TPP is a cofactor of two enzymes involved with the citric acid cycle. These enzymes bind and decarboxylate alpha-keto acids and facilitate the transfer of the aldehydes to enzyme CoA.

• Pyruvate dehydrogenase is part of the pyruvate dehydrogenase complex that converts pyruvate into acetyl-CoA. Acetyl-CoA provides 2-carbon units to the citric acid cycle.
• Alpha-keto glutarate dehydrogenase is part of the alpha-keto glutarate dehydrogenase complex that converts alpha-keto glutarate into succinyl-CoA. Succinyl CoA is converted into succinate.

The glycolytic pathway is the primary route for sugar degradation. However, an alternative pathway called the pentose phosphate pathway (hexose monophosphate shunt) also exists in cells. Oxidation of sugars through this pathway leads to the reduction of NADP to NADPH and the formation of D-ribose. NADPH is an important reducing agent for a number of metabolic processes and D-ribose is needed for nucleic acid synthesis. The TPP containing enzyme transketolase converts sugars formed in the pentose phosphate pathway back into sugars that can be metabolized by the glycolytic pathway. Hence, transketolase acts to bridge the two pathways and increases the efficiency of energy utilization.

Amino Acid Synthesis: TPP containing enzymes are required for the synthesis of the three amino acids; valine, isoleucine and leucine. All three of these amino acids are synthesized from pyruvate. TPP-enzyme bound acetyl groups are transferred directly into the synthetic pathways for valine and isoleucine. The acetyl group used to synthesize leucine is first transferred from a TPP-enzyme to acetyl-CoA and then into the leucine synthetic pathway.

Chemical Attributes of Thiamine that make it a Useful Serum-Free Medium Supplement:
Thiamine is a substituted pyrimidine linked to a substituted thiazole by a methylene group. It is generally added to cell culture as thiamine-2HCl.

• Formula C₁₂H₁₈Cl₂N₄OS
• Molecular Weight = 337.28
• Very water soluble
• Unstable in aqueous solutions above pH 5.5.
• May react with other media components-pyridoxal, iron, pyruvate (other alpha keto acids), and free radicals.
• Oxidizes to thiochrome.

The metabolically active form of thiamine is the diphosphate. It is formed by the enzymatic replacement of the hydroxyl group of the thiazole 5-hydroxyethyl side chain with pyrophosphate. Its biochemical activity is mediated by the thiazole moiety. The reactive chemistry of thiamine and thiamine pyrophosphate should be similar in solution.

Possible Chemical Instability in Cell Culture: Thiamine is not a very stable molecule. It can participate in a wide range of reactions that result in its destruction.

The metabolically relevant reaction site of thiamine and TPP is carbon 2 of the thiazole ring. It is situated between nitrogen and sulfur atoms. The proton on this carbon is acidic and at pH above 5.5 it dissociates to form a carbanion which undergoes nucleophilic addition to carbonyl groups. In the presence of alpha keto acids, such as pyruvate or alpha keto glutarate, thiamine may form thiamine:keto-acid adducts. The redox and pH conditions in the media will affect the degradation of these keto acids into aldehydes (reducing conditions) or acids (oxidizing conditions) and carbon dioxide.

Thiamine and TPP contain a primary amine on the pyrimidine moiety. This primary amine may form a Schiff base with pyridoxal or other aldehydes. In the presence of transition metals, such as iron, Schiff bases may cause the destruction of thiamine.

Oxidizing and reducing agents can destroy thiamine. One oxidation product is thiochrome. This molecule that may be detected by its fluorescence under uv light. Hypochlorite, sulfites and SO₂ degrade thiamine. Sulfur dioxide reacts irreversibly with thiamine to yield pyrimidine sulfonic acid and 4-methylhydroxyethyl thiazole. These molecules can form in oxidatively stressed media.

Mechanism of Action: TPP is a coenzyme for two types of enzymes, alpha-keto acid dehydrogenases and transketolases, both of which cleave a C-C bond adjacent to a carbonyl group releasing either carbon dioxide or an aldehyde. The resulting product is then transferred to an acceptor molecule.

Alpha-keto acid dehydrogenases decarboxylate alpha-keto acids. The decarboxylation product is then transferred to coenzyme A (CoA).

Transketolase cleaves the C-C bond adjacent to the carbonyl group of an alpha-keto sugar to give an activated glycoaldehyde. The glycoaldehyde is then combined with an aldose to give a new ketose.