Glutathione in Cell Culture

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

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

Glutathione is a water soluble tripeptide that possesses active thiol chemistry and which is very important for cell growth and viability. Whether cells benefit from the addition of exogenous glutathione is dependent upon the situation. Under optimal conditions cells manufacture required glutathione. However, the ability of cells to produce optimal levels of glutathione is determined by nutritional factors and the condition of the cells.

Some, but not all, early cell culture media contain glutathione. The early media M199; McCoy's 5A Modified Medium; and Waymouth Medium MB contain glutathione. Waymouth Medium MB was developed as a defined medium. Medium 199's derivative CMRL-1066 Medium contains glutathione. CMRL-1066 was designed as a chemically defined medium and it contains 200 times more glutathione than Medium 199. NCTC Medium developed in the early 1960s also contains high levels of glutathione. RPMI-1640 developed for leukocyte culture contains glutathione. Williams Medium E contains glutathione. A number of media used as the basis for development of proprietary media useful in biomanufacturing and tissue engineering do not contain glutathione in their formulae. These include: Dulbecco's Modified Eagle's Medium (DMEM); Nutrient Mixture, Ham's F-12; DMEM/Ham's Nutrient Mixture F-12 (50:50); and Iscove's Modified Dulbecco's Medium (IMDM). Serum-Free/Protein Free Hybridoma Medium does not contain added glutathione, whereas H-Y Medium (Hybri-Max®) does.

Efforts to develop serum-free and protein free media useful for biomanufacturing and tissue engineering have revealed an ill defined requirement for exogenous glutathione. Part of this mystery is resolved by understanding how the availability of other components of media may affect the cells requirement for glutathione. Important factors include the level of oxidative stress, the ability of cell to utilize cysteine and cysteine equivalents and cell phenotype. For a more complete discussion of glutathione as a cell culture additive go to Sigma's Media Expert.

Primary Functions of Glutathione in Cell Culture Systems:

Glutathione protects cells from oxidative stress and contributes to a favorable redox environment both inside and outside the cell. It provides reducing equivalents to regenerate sulfhydryl compounds and antioxidants, and to convert peroxides to water or alcohols. Oxidized glutathione is rapidly reduced by glutathione reductase and under normal in vivo or in serum conditions very little exists as oxidized glutathione.

Glutathione is the preferred substrate of a number of seleno-proteins that detoxify the intra- and extra- cellular environments by converting hydrogen peroxide to water and organic hydroperoxides into water and organic alcohols. If not removed, these peroxides and hydroperoxides ultimately form reactive hydroxyl, peroxyl and alkoxyl radicals.

Glutathione reduces cystine and cysteine mixed disulfides to form cysteine. Hence it functions to maintain cysteine in its reduced form and to mobilize it when it exists as a mixed disulfide. This keeps cysteine available as a substrate for the formation of proteins and glutathione itself.

Glutathione regenerates ascorbate, an important vitamin involved in copper mobilization, cell attachment, amino acid catabolism, and anti-oxidation. Vitamin E is a primary membrane associated antioxidant of the cell. It functions by providing hydrogen to convert lipid peroxyl radicals to hydroperoxides. Vitamin E is regenerated by ascorbate, and ascorbate is subsequently regenerated by glutathione. Hence glutathione indirectly facilitates anti-oxidation activity in cell membranes.

Reduced glutathione is an efficient chelator of cuprous copper. It has been shown that when cuprous copper is added to a solution containing ascorbate that the rate of ascorbate autoxidation accelerates. Glutathione appears to inhibit the copper mediated autoxidation of ascorbate.

Glutathione functions through glutathione S-transferases to detoxify reactive aldehydes created during the process of lipid peroxidation and/or glyoxidation.

Glutathione may serve as a non-toxic and stable reservoir of cysteine for protein synthesis.

Glutathione keeps intracellular protein thiol groups reduced. This facilitates the secretion of proteins.

Certain cells use a special amino acid membrane transport system that depends upon the enzyme gamma-glutamyl transpeptidase. Glutathione is a preferred substrate of this enzyme. Hence, glutathione facilitates the transport of other amino acids into the cell.

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

Glutathione is a tri-peptide with a molecular weight of 307.33.  It is composed of glutamate, cysteine and glycine amino acid residues. Cysteine is linked to glutamate through the gamma carbon of the glutamate side chain. Glutathione contains a thiol group that participates in redox reactions. It can accept or donate single electron equivalents and exists as reduced glutathione, oxidized di-glutathione, mixed disulfides and metal coordinated dithiols.

Reduced glutathione is several orders of magnitude more stable than ascorbate or cysteine to autoxidation. It non-enzymatically reduces other molecules such as dehydroascorbate, alpha-tocopherol, cystine and protein disulfides. Oxidized glutathione is reduced enzymatically by the enzyme glutathione reductase (EC and receives its electron equivalents from NADPH.

In vitro, glutathione generally exists in the presence of other thiol species such as those mentioned above. Because of its propensity to reduce these other molecules, it actually has a short half-life in the reduced form and must be continually reduced enzymatically. Glutathione is present in serum primarily in the reduced form. In the absence of serum or cells, it is likely to exist primarily in the oxidized form.

Glutathione 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 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 eucaryotic mammalian cell culturing system visit the Media Expert for helpful guidance.