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Riboflavin in Cell Culture

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

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

Riboflavin is a water soluble vitamin that functions as a prosthetic group of flavoproteins. It binds important macromolecules such as serum albumin in cell culture formulations. Most media contain some level of riboflavin in their basal formulations. L-15 Medium is an exception.

The concentration of riboflavin in basal cell culture media formulations ranges from lows of 0.01 µM in MCDB medium, 131; and 0.027 µM in Ames' Medium; CMRL-1066 Medium; and Medium 199 to highs of 1.33 µM and 2.66 µM in Fischer's Medium and Waymouth Medium MB, respectively. NCTC Media contains 0.067 µM riboflavin. The concentration of riboflavin in Basal Medium Eagle (BME); Minimum Essential Medium Eagle (EMEM) and Williams Medium E is 0.27 µM, which is similar to 0.30 µM found in MCDB media, 105, 110 and 201.

Nutrient Mixture, Ham's F-10 contains 1.0 µM of riboflavin, which is essentially the same concentration present in Dulbecco's Modified Eagle's Medium (DMEM); Iscove's Modified Dulbecco's Medium (IMDM); and H-Y Medium (Hybri-Max®). Nutrient Mixture, Ham's F-12 contains 0.1 µM of riboflavin, which is essentially the same concentration as MCDB Media, 151 and 301; F-12 Coon's Modification; F-12 Coon's Modification; Swim's S-77 Medium; and Serum-Free/Protein Free Hybridoma Medium.

The concentration of riboflavin in the popular DMEM/Ham's Nutrient Mixture F-12 (50:50) used as a starting formulation for development of proprietary serum-free, or protein-free cell culture media for Chinese Hamster Ovary (CHO) cell based biomanufacturing of heterologous proteins is 0.59 µM. This is only slightly higher than the 0.53 µM found in BGJb Medium Fitton-Jackson Modification; Click's Medium; Glascow Modified Eagle's Medium (GMEM); McCoy's 5A Modified Medium and RPMI-1640.

Serum-free and protein-free cell culture systems, especially those developed for biomanufacturing of heterologous proteins and tissue engineering appear to benefit from the addition of exogenous riboflavin in a concentration range from 0.5 to 1.0 µM. The actual effective concentration may depend upon the delivery form, free or bound, and the storage and use conditions of the medium. Riboflavin is sensitive to light and forms toxic complexes with certain amino acids and metals. This may explain the range of concentrations of riboflavin used in traditional media, where variables such as serum and protein use and storage of the medium affect riboflavin.

The chemistry and bioavailability of riboflavin in cell culture plays an important role in the stability and utility of media used for biomanufacturing of heterologous proteins and tissue engineering. For a more complete discussion of riboflavin as a cell culture component, visit our Media Expert.

Primary Functions of Riboflavin in Cell Culture Systems:

Riboflavin is important for a wide range of metabolic reactions involving oxidases and dehydrogenases. As a prosthetic group of flavoproteins, it facilitates the transfer of electrons and hydrogen atoms towards the reduction of oxygen. The following is a partial list of some important flavoproteins and their metabolic roles:

Energy Production:

Dihydrolipoyl dehydrogenase (EC oxidizes enzyme bound lipoic acid in the pyruvate dehydrogenase complex and transfers two hydrogens to NAD.

Succinic dehydrogenase (EC derives two hydrogens from the conversion of succinate to fumarate. These electron equivalents are subsequently supplied to the electron transport chain.

Vitamin Metabolism:

  • Pyridoxine phosphate oxidase (EC converts pyridoxamine phosphate and pyridoxine phosphate to pyridoxal phosphate.
  • Aldehyde oxidases use FAD to oxidize aldehydes, such as pyridoxal and retinal to their acid forms.
  • Methylene tetrahydrofolate reductase (EC or (EC is a key FAD containing enzyme in folate metabolism that converts 5,10 methylene tetrahydrofolate to 5-methyl tetrahydrofolate.


  • Glutathione reductase (EC, a FAD enzyme, reduces glutathione and helps maintain the anti-oxidation potential of the cell.
  • Xanthine oxidase (EC, a FAD enzyme, converts hypoxanthine and xanthine into uric acid. Uric acid is an important antioxidant in vivo.

Fatty Acid Metabolism

The fatty acyl CoA dehydrogenases are FAD containing enzymes that desaturate fatty acids. Desaturation is a very important step in the synthesis of fatty acids such as oleic, linoleic, linolenic and arachidonic acid.

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

Riboflavin Forms:

Riboflavin exits in three major forms:

  • Non-phosphorylated riboflavin is not incorporated into proteins as a prosthetic group. However, it is the vitamin form most frequently used in medium formulations (except Liebovitz L-15), and it is found in the intestines of animals.
  • Riboflavin-5’-phosphate, FMN, functions as a prosthetic group in some flavoproteins. In vivo, intestinal enterocytes use flavokinase to convert non-phosphorylated riboflavin to FMN. FMN is transported to the liver primarily on albumin. Cell culture systems supplemented with sera will receive some riboflavin in the form of FMN bound to albumin and some as FMN incorporated into flavoproteins.
  • Flavin adenine dinucleotide, FAD: Riboflavin delivered to the liver as FMN is incorporated into flavoproteins or further metabolized to FAD and then incorporated into flavoproteins. Riboflavin is then transported to other tissues primarily as FMN and FAD flavoproteins. Cell culture systems supplemented with sera receive some riboflavin in the form of FAD incorporated into flavoproteins.

All three forms of riboflavin can accept and donate electrons and the electron-equivalent, hydrogen. This is the basis for their ability to facilitate electron transfer when incorporated into flavoproteins. Riboflavin is present in cells almost exclusively as the mono-phosphate and adenine dinucleotide. Approximately, 70-90% exists as the flavin adenine dinucleotide (FAD). The ability of cells to grow in serum-free and protein-free media may be dependent upon their ability to metabolize riboflavin into FMN and FAD forms.

General features of non-phosphorylated riboflavin :

  • Chemical name: 9, D-ribitol, 6,7 dimethyl, isoalloxazine, C17H20N4O6, mol. wt. 376.4.
  • Neutral solutions of riboflavin are greenish-yellow and display yellowish green fluorescence with a maximum intensity at 565 nm. It has very little optical rotation activity at neutral pH.
  • Isoelectric point is approximately 6.
  • Dissociation constants at Ka = 6.3 X 10-12 and Ka = 0.5 X 10-5.
  • Slighty soluble in water. One g dissolves in 3-15 L water, depending on the crystal structure. It is less soluble in alcohol than in water (4.5 mg in 100 ml of absolute ethanol at 27 °C). Riboflavin is very soluble in dilute alkalies, but is unstable. A 10 mg/ml solution in 0.1 M NaOH forms a clear yellow to orange solution.
  • Non-dialyzable when it is bound to albumin.
  • Can be released from albumin with solvents and boiling.
  • At neutral pH, tryptophan and tyrosine can form stable charge-transfer complexes with riboflavin.
  • The isoalloxazine moiety of riboflavin chelates metals such as cadmium, cobalt, copper, iron, molybdenum, manganese, nickel, silver, and zinc. Some bound metals are easily oxidized and reduced and play a role in the formation of free radicals.
  • Stable in neutral solutions in the absence of light. Stable to air and normal oxidizing agents, and resistant to oxidation by chlorine gas, hydrogen peroxide (except when ferrous iron is also present), nitrous acid and hypochlorous acid.

General features of FMN:

  • Non-dialyzable when bound to albumin.
  • Can be release from albumin with solvents and boiling
  • More soluble in media than the non-phosphorylated form. Solubility at pH 6.9 is >100 mg/mL.
  • More sensitive to light than the non-phosphorylated form.
  • Much more light reactive than non-phosphorylated riboflavin

General features of FAD:

The most abundant form in vivo, but is generally associated with carrier molecules or flavoproteins.

Riboflavin Photolysis:

All three forms of riboflavin can be degraded by light. In cell culture, the primary concern is the breakdown of the non-phosphorylated and FMN forms.

  • Riboflavin decomposes in the presence of light at wavelengths below 500 nm, especially in the range from 350-400 nm. It is extremely unstable in sunlight and can be destroyed in just hours. The rate of destruction is even greater at elevated temperatures.
  • At neutral pH and in the presence of oxygen, the primary break-down products of riboflavin are:
    • Lumichrome, (6,7 dimethyl-alloxazine)
    • The ribityl side chain degrades into aldehydes and acids, such as acetaldehyde, formaldehyde, formic acid, etc

Riboflavin Complexes and Cell Toxicity:

Riboflavin in cell culture media complexes with various metals and amino acids. This is important because some of these complexes increase the production of toxic byproducts when exposed to light.

  • Hydrogen peroxide is formed when riboflavin is degraded by light in the presence of tryptophan or tyrosine. Hydrogen peroxide is also formed by riboflavin, but to a lesser extent, when these two amino acids are absent from the medium.
  • The riboflavin:tryptophan complex always leads to toxicity. In addition to hydrogen peroxide, this complex can generate singlet oxygen species that oxidize other amino acids such as histidine, methionine, and tyrosine resulting in additional toxic materials.

Concentrations of H2O2 as low as 1 mg/mL have been shown to be lethal to mammalian cells. H2O2 disrupts cell replication; it causes DNA and RNA damage; base destruction; single-strand breaks, double-strand breaks; cross-linkage, and sister chromatid exchanges. The presence of ferrous or cuprous ions in the media will promote the formation of hydroxyl radicals leading to the initiation of lipid peroxidation.

Riboflavin 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.

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