Importance and uses of riboflavin in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell, cultures
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.
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:
Dihydrolipoyl dehydrogenase (EC 126.96.36.199) oxidizes enzyme bound lipoic acid in the pyruvate dehydrogenase complex and transfers two hydrogens to NAD.
Succinic dehydrogenase (EC 188.8.131.52) derives two hydrogens from the conversion of succinate to fumarate. These electron equivalents are subsequently supplied to the electron transport chain.
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.
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.
The most abundant form in vivo, but is generally associated with carrier molecules or flavoproteins.
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 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.
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.
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.