Importance and uses of the B6 vitamins, pyridoxal and pyridoxine, in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell, cultures
Vitamin B6, in its various forms, is an essential additive to cell culture. It is a component of the basal medium formulation. Vitamin B6 has traditionally been added to cell culture media formulations as the aldehyde, pyridoxal; the alcohol, pyridoxine; or both the aldehyde and alcohol. One exception to this is BGJb Medium, Fitton-Jackson Modification which contains pyridoxal-5-phosphate. The form of Vitamin B6 in basal media is important because the aldehydes, pyridoxal and pyridoxal phosphate, are chemically reactive with primary amines. Any media which contains pyridoxal or pyridoxal phosphate is chemically unstable. Chemical instability is especially important in serum-free or protein-free media used for commercial applications, such as biomanufacturing of heterologous proteins and tissue engineering.
The following media contain pyridoxal alone or in combination with pyridoxine:
Media containing pyridoxal alone include: Ames' Medium; Basal Medium Eagle (BME); Click's Medium; Fischer's Medium; Glascow Modified Eagle's Medium (GMEM); Iscove's Modified Dulbecco's Medium (IMDM); Minimum Essential Medium Eagle (EMEM); Swim's S-77 Medium; Williams Medium E and various proprietary media. The original Dulbecco's Modified Eagle's Medium (DMEM) also contains pyridoxal, but several cell culture media manufacturers offer DMEM with pyridoxine substituted for pyridoxal.
Media that contain both pyridoxal and pyridoxine include: CMRL-1066 Medium; DMEM/Ham's Nutrient Mixture F-12 (50:50); H-Y Medium (Hybri-Max®); McCoy's 5A Modified Medium; Medium 199; and NCTC Medium.
Cell culture media that contain pyridoxine alone include: F-12 Coon's Modification; L-15 Medium; MCDB Media series; Nutrient Mixture, Ham's F-10; Nutrient Mixture, 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; and various proprietary media. These media benefit from improved stability, but whether a specific cell can utilize pyridoxine in culture needs to be determined.
The chemistry and bioavailability of Vitamin B6 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 Vitamin B6 as a cell culture component, visit our Media Expert.
Vitamin B6 is involved in a wide range of biochemical reactions that affect cell processes:
Pyridoxal phoshate (PLP), an adehyde, is the active coenzyme in almost all B6 containing enzymes. The broad spectrum of biochemical reactions catalyzed by PLP-enzymes results from the formation of Schiff bases between the vitamin aldehyde and the substrate primary amines. Since all amino acids contain at least one primary amine, the Schiff base mechanism of action makes PLP-enzymes important in their metabolism.
The following are some reactions mediated by the Schiff base PLP-enzymes.
The vitamin B6 family includes pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their phosphorylated counterparts; pyridoxine phosphate (PNP), pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP). The vitamin is not synthesized in animal cells. However, mammalian cells can interconvert it. A series of phosphorylase, oxidase, and kinase enzymes effect the in vivo inter-conversion of pyridoxine and its derivatives.
In vivo, B6 compounds exist primarily as phosphorylated derivatives. In vitro, B6 compounds are provided in the media formulations as either pyridoxal or pyridoxine. The addition of serum or albumin to cell cultures may also provide pyridoxal and pyridoxal phosphate.
The aldehyde forms of vitamin B6, PL and PLP, can non-enzymatically break down primary amine containing molecules, such as amino acids, and may generate toxic by-products. This non-enzymatic degradation of amino acids is enhanced by transition metals that are chelated by the Schiff base.
Similar to the PLP-enzyme mediated reactions, pyridoxal:metal:amino acid Schiff base complexes participate in several types of non-enzymatic reactions:
In addition to the loss of amino acids due to these reactions, toxic by-products are often formed.
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.