Selenium in Cell Culture

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 diverse antioxidant functions and 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 is 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.

Cytosolic glutathione peroxidase was the first enzyme recognized as a selenoprotein. It acts as a catalyst in the reduction of hydrogen peroxide and a wider variety of organic hydroperoxides by glutathione. Glutathione serves as a cofactor, supplying the electrons for the reductive reaction. Other sulfur compounds that may also be utilized as cofactors of glutathione peroxidases and selenoprotein P include 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 accounts for 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 outer surface 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.

Which cell culture media formulations contain selenium?

Selenium is an essential mineral in cell culture that was not recognized in formulations developed prior to 1976 because sufficient quantities of the mineral were present in the serum or in the water used to prepare media. Consequently, selenium is absent from certain basal media formulations (Table 1).

Table 1. Selenium-free Cell Culture Media Formulations

Ames’ Medium	F-12, Coon’s Modification	McCoy's 5A Modified Medium
Basal Medium Eagle (BME)	Fischer’s Medium	Medium 199
BGJb Medium	Glasgow Minimum Essential Medium (GMEM)	NCTC Medium
BGJb/Fitton-Jackson Modification	Ham’s F-10 Nutrient Mixture	RPMI-1640
Click’s EHAA Medium	Ham’s F-12 Nutrient Mixture	Select Serum-Free/Protein-Free Hybridoma Media
CMRL-1066 Medium	Ham’s F-12K Kaighn’s Modification	Swim's S-77 Medium
Dulbecco's Modified Eagle's Medium (DMEM)	H-Y Medium (Hybri-Max®)	Waymouth Medium MB
DMEM/F-12 Ham’s Nutrient Mixture (50:50)	L-15 Medium (Leibovitz)	Williams’ Medium E
Eagle’s Minimum Essential Medium (EMEM)		Various proprietary media

Table 2. Select Serum-free/Protein-free Hybridoma Medium

Iscove's Modified Dulbecco's Medium (IMDM)	MCDB 131 Medium	MCDB 302 Medium
MCDB 105 Medium	MCDB 153 Medium	select Serum-free/Protein-free Hydbridoma Medium
MCDB 110 Medium	MCDB 201 Medium	DMEM (D0547)

In 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 respective modifications to proprietary serum-free or protein-free commercial media useful for recombinant heterologous protein production, including monoclonal antibody (Mab) manufacturing.

Selenium is chemically reactive in solution and can participate in oxidation/reduction (redox) and free radical reactions. Consequently, the simple inclusion of selenium in a basal formulation may lead to toxic effects. In some instances, selenium is now added to media based on the above formulations, and it is a component of the supplements ITS (insulin-transferrin-selenium), SPIT (sodium pyruvate insulin transferrin), and SPITE (sodium pyruvate-insulin-transferrin-ethanolamine) (see products below).

Primary Functions of Selenium in Cell Culture Systems

Whether selenium is added to cell culture media 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).

Organic and Inorganic Forms of Selenium in Cell Culture

Inorganic Forms of Selenium

• Selenium, Se;
• Selenium Dioxide, SeO₂;
• Selenite, SeO₃^2-

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, 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 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.