Ferric and Ferrous Iron in Cell Culture

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

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

Iron is an essential transition metal in cell culture. It has both beneficial and toxic properties. Consequently, the management of iron levels and delivery are a major challenge. The natural physiological delivery of iron to cells in culture is mediated by transferrin when serum is used. The development of serum-free, animal-protein- and protein-free media has greatly increased the potential for iron toxicity in culture. This is especially critical in biomanufacturing and tissue engineering where iron mediated oxidative and carbonyl stress can alter the chemistry of a cell culture product.

The classical and commercially available media can be divided roughly into three groups: those that do not have iron in the basal formulation: Ames' Medium; Basal Medium Eagle (BME); BGJb Medium Fitton-Jackson Modification; Click's Medium; CMRL-1066 Medium: Fischer's Medium; Iscove's Modified Dulbecco's Medium (IMDM); L-15; McCoy's 5A Modified Medium; Medium 199; RPMI-1640; Swim's S-77 Medium; Waymouth Medium MB; those that contain ferric nitrate: Dulbecco's Modified Eagle's Medium (DMEM); Glascow Modified Eagle's Medium (GMEM); H-Y Medium (Hybri-Max^®); Medium 199; and Williams Medium E; and those that contain ferrous sulfate (generally based on Ham's Nutrient Mixtures): F-12 Coon's Modification; Nutrient Mixture, Ham's F-10; Nutrient Mixture, Ham's F-12; Nutrient Mixture, Ham's F-12 Kaighn's Modification (F12K); MCDB Media; and Serum-Free/Protein Free Hybridoma Medium.

DMEM/Ham's Nutrient Mixture F-12 (50:50) contains both ferric nitrate and ferrous sulfate.

A number of media used as the basis for development of proprietary media useful in biomanufacturing and tissue engineering do not contain iron in their formulae or have been re-engineered specifically to manage iron delivery and toxicity. Proprietaty media developed from Dulbecco's Modified Eagle's Medium (DMEM); Nutrient Mixture, Ham's F-12; and DMEM/Ham's Nutrient Mixture F-12 (50:50) or any other inorganic iron supplemented media typically have the iron salts removed and replaced by a chelated iron. Other important basal media for proprietary media development such as Iscove's Modified Dulbecco's Medium (IMDM) are generally supplemented with a chelated iron.

The management of iron delivery is one of the most complex, yet important, requirements for the development of a suitable cell culture system for biomanufacturing and tissue engineering. Improper iron management affects not only the cell, but also the quality of the cell product. For a more complete discussion of chelated-iron as a cell culture additive, visit our Media Expert^®.

Primary Functions of Iron in Cell Culture Systems:

Iron is essential for cell respiration and metabolism. Without iron cells stop growing and eventually die. Iron can undergo univalent redox reactions. Its oxidized and reduced forms are referred to as ferric and ferrous iron, respectively.

• Hemoglobin is the quantitatively predominant heme containing protein in mammals. Its primary function is to bind, transport, and release molecular oxygen. Hemoglobin bound iron remains in the ferrous state during oxygen binding, transport and release. When hemoglobin bound iron is oxidized to ferric iron, it cannot transport oxygen. Oxidized hemoglobin is called methemoglobin.
• Cytochromes are a group of heme containing proteins located in the mitochondria. Their function is the sequential transfer of electrons along the respiratory chain leading to reduction of oxygen to water, and the storage of energy in the form of ATP. The transfer of electrons by cytochromes involves oxidation and reduction cycling of iron.
• Iron exists in a non-heme form as part of succinate dehydrogenase (EC 1.3.99.1), an enzyme that catalyses the formation of fumarate.
• As an enzyme cofactor, iron protects cells from oxidative damage. It exists as a heme component of enzymes such as catalase and peroxidase that catalyze the conversion of peroxides to water.

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

The design of a stable and useful medium requires an understanding of the chemistry of iron. The management of this metal is a critical success factor in cell culture.

Iron exists in both the ferrous, and ferric state in physiological solutions. It is frequently added to cell culture media as a nitrate or sulfate salt. In solution, it undergoes redox cycling depending upon the medium’s composition of oxidizing and reducing agents, and its exposure to these agents. The susceptibility of iron to the medium’s redox potential depends upon the molecules that complex it. Iron complexes can form with bio-molecules such as amino acids, nucleotides, physiological chelators, and proteins. The specific complexes are very important because they determine whether the iron is available to participate in cell growth, to catalyze toxic reactions or becomes non-available to the system. Free or ineffectively sequestered iron can be very toxic to cells. Properly complexed iron is available to support cell life and is essential to the cell culture system.

The most appropriate extracellular physiological complexes of iron are with ferritin and transferrin. Ferritin is the primary storage molecule for iron.

Ferric Iron:

Ferric iron is the stable oxidative state of iron in aerobic conditions, and the normal oxidative state used by cells. In a solution at physiologic pH, ferric iron that is not bound by a chelator or carrier molecule will form ferric hydroxide complexes that are virtually insoluble. The chemistry is quite complex. However, it is not necessary to fully understand it to appreciate that iron may be lost due to the formation of these complexes. Ferric iron can be reduced to ferrous iron by strong reducing agents in the medium. Iron reducing agents of concern in cell culture include the superoxide radical and ascorbate. A widely studied cellular reductant and frequently used media component is ascorbate (Vitamin C). Ascorbate can reduce ferric:EDTA to ferrous:EDTA. Ferrrous:EDTA can catalyze the formation of hydroxyl free radicals through Fenton chemistry.

Both the loss of ferric iron due to precipitation and its reduction to ferrous iron are undesirable events in the extracellular milieu. Both of these occurrences are avoided when transferrin is included in the culture system. In view of the above facts, one should strongly consider whether adding iron salts directly to a medium is appropriate. This question becomes increasingly more important in serum-free medium where the protective qualities of serum are absent.

Ferrous Iron:

Ferric iron can be reduced to ferrous iron in cell culture media. Iron in the ferrous oxidative state that is free to participate in Fenton chemistry is a major source of oxidative stress in media. The reaction involves the ferrous iron catalyzed conversion of hydrogen peroxide into a hydroxide ion and a hydroxyl free radical with the concurrent oxidation of ferrous iron to ferric iron. This reaction is profoundly important in cell culture, because when it occurs, it creates the most damaging agent found in the cell culture system-the hydroxyl free radical. This radical will react with virtually any molecule in the medium or the cell. It is so reactive that it typically reacts very close to its site of formation. A major action of hydroxyl free radicals in cell culture involves the initiation of lipid peroxidation. Ferrous iron mediated lipid peroxidation can occur with iron in a number of chelated complexes: the ferrous iron can be chelated to phosphate, ADP, ATP, oxalate, and citrate.

Enzymes that catalyze the conversion of ferrous iron to ferric iron are ferroxidases. Ceruloplasmin, a serum protein, has a ferroxidase activity that accelerates the oxidation of ferritin bound ferrous iron into ferric iron and promotes its binding by transferrin. In the absence of ferroxidase activities, the conversion of ferrous iron to ferric iron is slow.

Ferrous and ferric iron will complex with thiols such as cysteine. The ferrous:cysteine complex can participate in the generation of hydroxyl free radicals through Fenton chemistry.

Iron Products that Enhance the Growth of Hybridoma, Chinese Hamster Ovary (CHO) and other Mammalian Eukaryotic Cells in Serum-free Cultures:

Our 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 eukaryotic mammalian cell culturing system visit the Media Expert^® for helpful guidance