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.