Vitamin E, tocopherol, in Cell Culture

Importance and uses of vitamin E in serum-free eucaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell cultures

Vitamin E, a Serum-Free Media Supplement, Useful In Biomanufacturing; Tissue Engineering and Specialty Media

Vitamin E (alpha-tocopherol) is an important cell membrane anti-oxidant that is unstable and poorly soluble in aqueous media. Traditionally it has been added to cell culture systems as part of the serum supplement. For these reasons most classic media do not have vitamin E in their basal formulation. These classic media include: Ames' Medium; Basal Medium Eagle (BME); Click's Medium; CMRL-1066 Medium: Dulbecco's Modified Eagle's Medium (DMEM); DMEM/Ham's Nutrient Mixture F-12 (50:50); F-12 Coon's Modification; Fischer's Medium; Glascow Modified Eagle's Medium (GMEM); Iscove's Modified Dulbecco's Medium (IMDM); L-15 Medium; McCoy's 5A Modified Medium; MCDB Media; Minimum Essential Medium, Eagle (EMEM); Nutrient Mixture, Ham's F-10; Nutrient Mixture, Ham's F-12; Nutrient Mixture Ham's F-12 Kaighn's Modification (F12K); RPMI-1640; Swim's S-77 Medium; Waymouth Medium MB and various proprietary and specialty media.

There are a some classic media that contain alpha-tocopherol. NCTC Medium and H-Y Medium (Hybri-Max®), which is 1/8th NCTC medium, contain 54 nM and 6.7 nM of vitamin E, respectively. Medium 199 and Williams Medium E contains 11.6 nM of vitamin E. Serum-Free/Protein Free Hybridoma Medium, a modification of Ham's F-12 medium contains 324 nM vitamin E and BGJb Medium Fitton-Jackson Modification contains the highest level of tocopherol at 2.16 µM.

The inclusion of vitamin E may provide some benefits when these media are used fresh. However the chemical instability and poor solubility of tocopherol probably result in the loss of significant amounts of the vitamin when it is aseptically processed and stored. For a more complete discussion of vitamin E as a cell culture media supplement go to Sigma's Media Expert.

Primary Functions of Vitamin E in Cell Culture Media

Aerobic cells use oxygen as the ultimate electron acceptor in respiration. In the process, oxygen is reduced to water. Oxygen is also involved in a complex series of reactions referred to as oxidative stress. A major consequence of oxidative stress is lipid peroxidation. Lipid peroxidation destroys cell membranes and causes cell death.

α-Tocopherol, Vitamin E, is an important lipid peroxidation antioxidant found in cell membranes. It acts as an antioxidant by donating a hydrogen atom to a lipid-peroxyl radical. In the process, the lipid-peroxyl radical becomes a lipid-hydroperoxide and α-tocopherol is oxidized to a free radical. As a free radical, tocopherol has three potential fates:

  • it can react with a second lipid-peroxyl radical and become a stable product;
  • it can react with another α-tocopherol free radical to form a dimer,
  • or it can be regenerated by a reducing agent such as ascorbate.

The first two situations lead to the depletion of α-tocopherol as a lipid antioxidant. The third situation maintains tocopherol as an active lipid antioxidant.

Chemical Attributes of Vitamin E that make it a Useful Serum-Free Media Supplement:

α-Tocopherol is a member of a class of compounds known as the tocopherols. Purified α-tocopherol exists as a slightly viscous, pale yellow oil, which is practically insoluble in water. Tocopherol slowly oxidizes in the presence of oxygen, and the rate of oxidation accelerates in the presence of light and ferric iron. α-Tocopherol acetate and α-tocopherol phosphate have been used as supplements in cell culture. α-Tocopherol acetate is more stable to light and oxidation than underivitized tocopherols, but it is also poorly soluble in water. α-Tocopherol phosphate may be slightly more soluble because of the ionic nature of the phosphate moiety. In each case, the poor solubility of tocopherol in water requires it to be complexed with another molecule such as a lipoprotein or added in a way that facilitates its rapid incorporation into a lipid moiety, such as the cell membrane.

In vivo, tocopherol is found primarily in cell membranes. In serum, such as FBS, it is bound to and transported primarily by lipoproteins. Its physiological concentration in serum is typically in the range from 18 to 32 mM.

Vitamin E Products that Enhance the Growth of Hybridoma, Chinese Hamster Ovary (CHO) and other Mammalian Eucaryotic Cells in Serum-free Cultures.

     

Sigma's Cell Culture Media Expert provides in depth discussion of this and other serum-free and protein-free media supplement(s). 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 eucaryotic mammalian cell culture media visit the Media Expert for helpful guidance.

Content for this page is provided by Dennis R. Conrad, Ph.D., a Life Science industry consultant with over 25 years of experience in the formulation and optimization of cell culture media. Dr. Conrad's email address is biomediaexpert@earthlink.net