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Biotin in Cell Culture

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

Biotin, both a Classic media and Serum-Free Media Supplement, Useful In Biomanufacturing, Tissue Engineering and Specialty Media:

Biotin is an important water soluble vitamin found in numerous media. These may include serum-free media used for biomanufacturing, tissue engineering, vaccine production and other commercially useful applications.

Biotin is found in many classic media including: Ames' Medium; Basal Medium Eagle (BME); BGJb Medium Fitton-Jackson Modification; CMRL-1066 Medium; DMEM/Ham's Nutrient Mixture F-12 (50:50); EMEM-alpha; F-12 Coon's Modification; Fischer's Medium; H-Y Medium (Hybri-Max®); Iscove's Modified Dulbecco's Medium (IMDM); McCoy's 5A Modified Medium; MCDB Medium; Medium 199; 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 specialty media.

It should be noted that culture media that lack biotin are generally based on Minimum Essential Medium Eagle (EMEM) and include: Click's Medium; Dulbecco's Modified Eagle's Medium (DMEM); Glascow Minimum Essential Medium (GMEM); and Swim's S-77 Medium. IMDM and EMEM-alpha are the biotin containing exceptions. L-12 Medium also lacks biotin.

Basal classic media used in the development of serum-free formulations for the biomanufacture of heterologous proteins and other commercial applications typically contain biotin either as part of the parent formulation or as a new addition to the proprietary or specialty medium.

Supplementation of media with biotin for serum-free culture of eukaryotic cells is complicated by the fact that biotin is susceptible to oxidative stress and inactivation by u.v. radiation. Hence the effective use of biotin in cell culture media requires an understanding of its chemistry. For a more complete discussion of biotin as a cell culture media supplement go to the Media Expert.

Primary Functions of Biotin in Cell Culture Media:

Biotin is an essential vitamin that is important for amino acid and energy metabolism, and fatty acid synthesis. It is a prosthetic group in four mammalian carboxylase families and facilitates the binding and transfer of carbon dioxide. The biotin containing enzymes are pyruvate carboxylase (EC, acetyl-CoA carboxylase (EC, methylcrotonyl-CoA carboxylase (EC, and proprionyl-CoA carboxylase (EC All four of these enzymes are ATP-driven. Pyruvate carboxylase and acetyl-CoA carboxylase are important for fatty acid synthesis. Methylcrotonyl-CoA carboxylase and proprionyl-CoA carboxylases are involved in the degradation of leucine and methionine, isoleucine and homocysteine, respectively.

Importance in vitro:

In the absence of biotin, mammalian cells cannot synthesize fatty acids. This makes these cells dependent upon external sources of fatty acids, including the palmitoyl fatty acid family. They cannot convert methionine, isoleucine, homocysteine or leucine into TCA cycle intermediates and they cannot convert excess pyruvate into fatty acids.

Biotin Protein Cycle:

Carboxylases are synthesized as apocarboxylases. Biotin is added to carboxylases by biotin holocarboxylase synthetases, such as biotin holocarboxylase synthetase (EC These synthetases attach biotin to the epsilon amino group of lysine and form the epsilon-N-biotinyl-L-lysyl amino acid residue. When carboxylase proteins are degraded, this amino acid that is also known as biocytin is released. Biotin can be released from biocytin by a ubiquitous mammalian cell enzyme called biotinidase (EC Biotinidases may have additional roles involving the transport and cell utilization of biotin.

Protein and Polyamine Biotinylation:

Biotin may play a role in the regulation of transcription and DNA repair. Under certain conditions, the enzyme biotinidase appears to function as a biotin transferase and transfer biotin to lysyl residues of histones and polyamines. Histones are important proteins that affect chromatin structure and access to DNA by regulatory and repair proteins. Biotinylation of histones may have a role in silencing of genes and cellular response to DNA damage. Polyamines are involved in important mitochondrial functions.

Biotin and histones outside the nucleus have some insulin-like activities. They increase the uptake of glucose and stimulate the synthesis of cGMP and formation of nitrous oxide. Biotin can induce glucokinase.

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


Vitamin H, CoEnzyme R

Biotin has a sulfur atom in its ring like thiamin and a transverse bond across the ring. Biotin has three asymmetric carbons and can exist as eight different isomers. Only the d-(+)-biotin, D-Biotin isomer is biologically active.

Formulation: C10H16N2O3S

Molecular Weight: 244.31

Isoelectric pH = 3.5

Biotin is:

  • water-soluble;
  • soluble in dilute alkali and hot water;
  • relatively stable at acidic and neutral pH range


Freely soluble in water

Precursor form of biotin, converted to biotin by biotinase.

Biotin Stability:

Biotin is a relatively stable vitamin under physiological conditions, but it may be susceptible to elements of oxidative stress. Peroxidized unsaturated fatty acids or exposure to ultraviolet radiation may inactivate biotin. Partial protection by vitamin E suggests that this inactivation probably occurs by fatty acids mediated free radical attack on biotin. The most susceptible site is probably the sulfur atom. Oxidation products of biotin are biotin sulfoxide and biotin sulfone. Other agents that may inactive biotin are formaldehyde, nitrous acid and choline.

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