Ascorbate in Cell Culture

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

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

Ascorbic acid is an important water soluble anti-oxidant found in numerous media. These may include media used for biomanufacturing, tissue engineering, vaccine production and other commercially useful applications.  Ascorbate is found in BGJb medium; CMRL-1066 medium; Nutrient Medium F-12 (Coon's modificaton); Hbyri-Max (R); McCoy's 5A; Medium 199; Alpha-EMEM; NCTC medium; Waymouth medium MB, William's medium E and various proprietary media. Supplementation of media with ascorbate for serum-free culture of eukaryotic cells is complicated by the fact that ascorbate is very unstable and rapidly oxidizes in aqueous systems.  Hence the effective use of ascorbate in cell culture requires an understanding of its chemistry. For a more complete discussion of ascorbate as a cell culture additive go to our Media Expert.

Primary Functions of Ascorbate in Cell Culture Systems:

Ascorbic acid (vitamin C) is an essential vitamin for the growth and maintenance of healthy cells in vivo and in vitro.

• Ascorbic acid is a water-soluble antioxidant that protects esterified and non-esterified unsaturated fatty acids from peroxidation. It converts lipid peroxyl radicals and alkoxyl radical into lipid hydroperoxides and lipid hydroxides, respectively. Hydroperoxides are relatively stable in the absence of iron and copper.
• Ascorbic acid regenerates membrane bound alpha-tocopherol (vitamin E) that has been oxidized by lipid peroxyl radicals and indirectly limits lipid peroxidation in cell membranes.
• Ascorbic acid regenerates urate from the urate radical. Urate is an important aqueous anti-oxidant.
• Ascorbic acid facilitates the mobilization of copper from serum ceruloplasmin into cells without producing hydrogen peroxide.
• Ascorbic acid is a cofactor for the enzyme prolyl hydroxylase (EC 1.14.11.2) that catalyzes the post translation hydroxylation of proline residues in nascent collagen and elastin molecules. Post-translation hydroxylation of these residues increases the degree of intra-molecular cross-linking of collagen and elastin and facilitates the development of extracellular matrices.
• Ascorbic acid is a hydroxylphenylpyruvic acid oxidase (EC 1.2.3.13) cofactor, an enzyme that facilitates the catabolism of amino acids phenylalanine and tyrosine to acetyl-CoA.

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

Ascorbic acid is a hexenoic sugar acid, gamma-lactone with an enediol structure at carbons 2 and 3. It can be reversibly oxidized and reduced by losing or gaining reducing equivalents from the oxygen atoms associated with carbons 2 and 3. Ascorbic acid, exists primarily as an ascorbate anion, at physiological pH.

Univalent Chemistry: Ascorbic acid is extremely unstable in vitro. It reduces other radicals including the hydroxyl radical, organic alkoxyl and peroxyl radicals, urate radical, tocopherol radical, and the ferric and cupric ions. It readily oxidizes to dehydro-ascorbate in a two step reaction. When the ascorbate anion loses one electron, it becomes an ascorbate radical, A·.

This ascorbate radical has several possible fates in vitro. It can

• loose another electron and become dehydro-ascorbate,
• gain an electron and proton to regenerate the ascorbate anion,
• react with another radical and form a stable compound, or
• two ascorbate radicals can dismutate to form one ascorbate anion and one dehydro-ascorbate molecule.

Univalent Chemistry:

Ascorbic acid is extremely unstable in vitro. It reduces other radicals including the hydroxyl radical, organic alkoxyl and peroxyl radicals, urate radical, tocopherol radical, and the ferric and cupric ions. It readily oxidizes to dehydro-ascorbate in a two step reaction. When the ascorbate anion loses one electron, it becomes an ascorbate radical, A·.

This ascorbate radical has several possible fates in vitro. It can

• loose another electron and become dehydro-ascorbate,
• gain an electron and proton to regenerate the ascorbate anion,
• react with another radical and form a stable compound, or
• two ascorbate radicals can dismutate to form one ascorbate anion and one dehydro-ascorbate molecule.

Loss of Ascorbate:

At pH 7.0, 37 °C, dehydro-ascorbate breaks down to 2,3-diketo-L-gulonic acid with a half-life of about 6.7 minutes. Superoxides react with dehydro-ascorbate to form oxalic and threonic acids. Hence, when reducing agents that are able to regenerate ascorbate from dehydro-ascorbate or ascorbate radical are missing from the system, ascorbate is lost.

Oxidative Stress:

In the presence of oxygen, under physiological conditions, the ascorbate anion does not have the redox potential to reduce oxygen to the superoxide radical. However, it is capable of reducing the superoxide radical formed by other means to hydrogen peroxide. Ascorbic acid also reduces the transition metals iron and copper. When superoxide radicals and iron or copper are present in a form that can undergo redox cycling, ascorbic acid facilitates the formation of hydroxyl free radicals. These free radicals cause extensive damage to a wide range of biomolecules and initiate lipid peroxidation.