Selenium in Cell Culture

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

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

Selenium is an essential metal in cell culture that was not recognized in formulations developed prior to 1976 because sufficient quantities of the metal were present in the supplements, such as serum or in the water used to prepare media. Consequently selenium is absent from the following basal formulations: Ames' Medium; Basal Medium Eagle (BME); BGJb Medium, Fitton-Jackson Modification; 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); H-Y Medium (Hybri-Max®); L-15 Medium; McCoy's 5A Modified Medium; Medium 199; Minimum Essential Medium, Eagle (EMEM); NCTC Medium; 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; Swim's S-77 Medium; Waymouth Medium MB; Williams Medium E and various proprietary media.

Selenium is included in the basal formulations of Iscove's Modified Dulbecco's Medium (IMDM); MCDB Media, 105, 110, 131, 153, 201 and 302; and in Serum-Free/Protein Free Hybridoma Medium and various proprietary media. Selenium is also added to Sigma's DMEM, D-0547.

It is worth noting that the basal formulations, DMEM/Ham's Nutrient Mixture F-12 (50:50) and Iscove's Modified Dulbecco's Medium (IMDM) are often supplemented with selenium as part of their modifications into proprietary serum-free or protein-free commercial media useful for recombinant heterologous protein, including monoclonal antibody (Mab), manufacturing.

Selenium is chemically reactive in solution and has the ability to participate in oxidation/reduction and free radical reactions. Consequently the simple inclusion of selenium in a basal formulation may lead to toxic effects. In some instance selenium is now added to media based on the above formulations, it is a component of the supplements ITS, SPIT, SPITE series of supplements (see products below). For a more complete discussion of riboflavin as a cell culture component go to Sigma's Media Expert.

Primary Functions of Selenium in Cell Culture Systems:

Selenium is an essential trace element for normal cell growth and development in vivo and in vitro. It is incorporated into enzymes that protect cells by reducing peroxides, organic hydroperoxides, and peroxynitrites to non-harmful species. Seleno-enzymes with various antioxidant functions, and different substrate specificities are localized inside, on and outside the cell, and together these enzymes provide a comprehensive range of defenses against oxidative damage.

Selenium is incorporated into glutathione and thioredoxin reductase(s), glutathione peroxidase(s), and selenoprotein P as the amino acid selenocysteine. Glutathione and thioredoxin reductase regenerate reduced glutathione and thioredoxin respectively. Reduced glutathione is the substrate for the peroxidases discussed below. Reduced thioredoxin is a substrate for thioredoxin peroxidases and a suppressor of apoptosis. The selenium-dependent glutathione peroxidase (GSHPx) family has at least four members that protect against the deleterious effects of peroxides and hydroperoxides. These include two cellular glutathione peroxidases, a plasma glutathione peroxidase, and a gastrointestinal tract associated glutathione peroxidase.

The cellular cytosolic glutathione peroxidase was the first enzyme recognized as a selenoprotein. It acts as a catalyst in the reduction of hydrogen peroxide and a variety of organic hydroperoxides by glutathione. Glutathione is utilized as a cofactor, supplying the electrons for the reductive reaction. Other sulfur compounds may also be utilized as cofactors of glutathione peroxidases and selenoprotein P, e.g., ascorbic acid, cysteine, cystamine, dithiothreitol, and mercaptoethanol.

Selenoprotein P (SeP) is an extracellular, multifunctional enzyme present in human plasma at a concentration of approximately 5 µg /mL. It represents approximately 50% of total selenium in human plasma. SeP may serve as the primary selenium transport and delivery protein to cells especially neuronal and immune cells. SeP also appears to bind to extracellular matrix materials and to the outside of cell membranes. In the presence of glutathione, cysteine or other sulfhydryl compounds, it can reduce lipid (fatty acid) hydroperoxides to corresponding alcohols. Hence, SeP protects cell membranes from lipid peroxidation

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

Whether selenium is added to cell culture as selenium; selenium dioxide or sodium selenite, it will change its form and oxidation state in response to the cell culture environment. It is important to understand the positive and negative effects that selenium species can have on the cell culture system in the context of their concentration(s) and form(s).

Forms of Selenium in Cell Culture:

Inorganic forms of Selenium:

  • Selenium;
  • Selenium Dioxide, SeO2;
  • Selenite, SeO3;

Organic forms of selenium:

  • Diselenide, RSeSeR or RSeSeR'; (R and R’ can be any organic moiety that links to selenium).
  • Selenodithiols, RS-Se-SR or RS-Se-SR'; (R and R’ can be any organic moiety that links to a sulfur).
  • Selenide anion (selenopersulfide), RSe; (R can be any organic moiety that links to selenium).

In vitro, selenium dioxide and selenite anions react with reduced thiol groups of glutathione, cysteine, DTT or 2-mercaptoethanol to form selenodithiols with the general formula RS-Se-SR'. Example compounds are seleno-diglutathione, GluS-Se-SGlu; seleno-dicysteine, CysS-Se-SCys; seleno-dithiothreitol; and seleno-dimercaptoethanol. Seleno-dithiols undergo oxidation/reduction to yield oxidized thiol compounds, such as GluSSGlu, and elemental selenium (Se). Elemental selenium is non-toxic and non-catalytic. Selenite reacts with reduced glutathione to produce superoxide. Further testing of inorganic selenium compounds demonstrated that the inorganic selenium compounds selenite and selenium dioxide, and the organic Diselenide of the composition RSeSeR reacts with thiols, such as glutathione, cysteine and dithiothreitol, and generated superoxide and hydrogen peroxide. Organic selenium of the RSeSeR or RSeSeR’ configuration can react with thiols to produce reduced selenide anions (selenopersulfide) represented as RSe-. Compounds of this class are toxic due to their catalytic acceleration of thiol oxidation that produces superoxide free radicals, hydrogen peroxide and the more toxic hydroxyl free radical.

In vitro, selenodithiols of the general formula RS-Se-SR' have biphasic effects on cell growth. Selenodithiols (and selenite) can inhibit protein synthesis and cell growth, they can also contribute to increased cell growth and protein production. The effect these selenium moieties have depends upon several factors. The most important of these appears to be the cell type and the concentration of the selenite, and the selenodithiols present. As a general rule, when the total concentration of selenium added to the cell culture system is above 1 µM, it may be inhibitory to cell growth and protein synthesis. Rapidly growing cells, such as tumor cells, are affected by selenodithiols at lower concentrations than normal cells.

Selenium 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.