Cysteine in Cell Culture

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

L-Cysteine and L-Cystine, Serum-Free Medium Supplements, Useful In Biomanufacturing; Tissue Engineering and Specialty Media

L-cysteine and/or its oxidation product, L-cystine, are essential amino acids in cell culture. L-cysteine is oxidized to L-cystine relatively easily, in vitro. The performance and stability of cell culture media is affected by the presence and ratios of the various forms of L-cysteine and L-cysteine equivalents. L-cysteine and L-cystine enter cells by different transporters which control the ability of specific cells to utilize L-cysteine in its various forms. Virtually all classical and serum-free media contain some form of L-cysteine. It may be added as L-cysteine, L-cystine, N-acetyl cysteine (NAC), L-cysteine mixed disulfides or in various peptides. In addition to its role in protein synthesis, L-cysteine is the functional activity of the tri-peptide glutathione. Its availability for glutathione synthesis is rate limiting.

L-cysteine is formulated into the following media: BGJb Medium Fitton-Jackson Modification; F-12 Coon's Modification; L-15 Medium; McCoy's 5A Modified Medium; MCDB Medium; Nutrient Mixtures, Ham's F-10; Nutrient Mixtures, Ham's F-12; Nutrient Mixture Ham's F-12 Kaighn's Modification (F12K); Serum-Free/Protein Free Hybridoma Medium and various proprietary media. When these media are stored as liquids, L-cysteine converts to L-cystine. Under extreme conditions L-cystine is further oxidized.

L-cystine is formulated directly into: Ames' Medium; Basal Medium Eagle (BME); Click's Medium; CMRL-1066 Medium: Dulbecco's Modified Eagle's Medium (DMEM); Fischer's Medium; Glascow Modified Eagle's Medium (GMEM); Iscove's Modified Dulbecco's Medium (IMDM); Minimum Essential Medium Eagle (EMEM); RPMI-1640 and various proprietary media.

Various ratios of both L-cysteine and L-cystine are formulated into DMEM/Ham's Nutrient Mixture F-12 (50:50); H-Y Medium (Hybri-Max^®); Medium 199; NCTC Medium; Waymouth Medium MB; Williams Medium E; and various proprietary media. Proprietary media used for bio-manufacturing and tissue engineering frequently contain additional L-cysteine equivalents such as N-acetylcysteine.

Supplementation of media with L-cysteine and L-cysteine equivalents for serum-free culture of eukaryotic cells is complicated by the fact that the R-chain(s) of L-cysteine and L-cystine contain sulfur atoms that participate in sulfur oxidation, reduction and radical chemistry and metal coordination chemistry. Hence the effective use of L-cysteine and L-cysteine equivalents in cell culture requires an understanding of its chemistry in the context of other media components.

Primary Functions of L-Cysteine and L-Cystine in Cell Culture Systems

Cystine and cysteine are nutritionally considered to be non-essential amino acids, because they are synthesized in the livers of mammals. However, for cell culture applications they are essential amino acids that must be supplied from an exogenous source. Effective supplementation is complicated by the fact that these two amino acids are non-enzymatically inter-convertible and unstable. Understanding their chemistry and the ways that cells utilize them are critical factors for designing a successful medium.

• Both cysteine and cystine exist as amino acid residues in proteins. Protein cystine residues are formed by oxidation of cysteine sulfhydryls after they have been incorporated. The formation and disruption of inter- and intra- protein disulfide bonds play important roles in determining secondary and tertiary structures.
• Cysteine serves a very important indirect role of protecting cells from oxidative stress. It is the rate-limiting amino acid used in the synthesis of the tri-peptide glutathione. Glutathione has the ability to oxidize dehydroascorbic acid to ascorbic acid, which is the primary aqueous antioxidant involved in blocking lipid peroxidation. It is also the substrate for the selenoprotein antioxidation enzymes. Glutathione is rapidly depleted and cells will die in the absence of L-cysteine or cysteine equivalents.
• Many cells metabolize cysteine to form hypotaurine and taurine. These two molecules help detoxify cell culture media by reacting with hydroxyl free radicals and hypochlorous acid, respectively. Taurine may also protect oxidatively damaged cells from death caused by calcium ion flux.
• Unpaired protein cysteine sulfhydryls can react with a free cysteine sulfhydryls to form mixed disulfides. These disulfides act as a storage and transport reservoir for cysteine equivalents. Albumin contains one non-paired cysteine sulfhydryl that can from a disulfide bond with a non-protein cysteine sulfhydryl. In this way, albumin can bind and shield the cysteine from other reactions in the medium. Protein-bound cysteine sulfhydryls can also react with other sulfhydryl molecules, such as glutathione, resulting in the formation of mixed disulfides.

Some possible fates of cystine and cysteine in cell culture include protein synthesis, auto-oxidation, mixed disulfides with proteins, synthesis of glutathione, and synthesis of Enzyme CoA.

Chemical Attributes of L-Cysteine and L-Cystine That Make it a Useful Serum-Free Medium Supplement

Cysteine has the empirical formula C₃H₇O₂NS and a molecular weight of 121.16. Cystine has the empirical formula C₆H₁₂O₄N₂S₂ and a molecular weight of 240.31. It is composed of two cysteine molecules joined by a disulfide bond. Cystine and cysteine are inter-convertible sulfur containing amino acids. The sulfur atoms bound to these amino acids provide sites for redox activity and electron transfer. Sulfur atoms can exist as free radicals.

In vitro, cysteine is freely soluble and exists initially as a neutral amino acid. It is unstable and undergoes non-enzymatic autoxidation. Ferric and/or cupric cations accelerate its autoxidation. The resulting reduced cations complex with the remaining cysteine (e.g. cysteine:ferrous complex) and catalyze the conversion of hydrogen peroxide into hydroxyl free radicals and sulfide free radicals.

Cysteine and Cystine Radical Chemistry:

Oxidation of cysteine is greatly augmented by the transition metals copper and iron. Univalent oxidation of cysteine generates the thiyl radical of cysteine. The cysteine thiyl free radical can participate in reactions with dimolecular oxygen, cysteine, another cysteine thiyl radical or other free radicals. Reactions with oxygen or cysteine generate other free radicals and propagate the oxidative process. Reaction with another thiyl free radical terminates the free radical propagation. Reaction with another cysteine thiyl free radical will form cystine. Cystine is susceptible to further oxidation to cysteic acid and other oxidative products. The cystine radical can react with hydrogen peroxide to form the hydroxyl free radical. The oxidation reactions of cysteine and cystine free radicals are not limited to those described above.

Copper may affect the solubility of cysteine and cystine. Cuprous copper forms cuprous mercaptide of cysteine and cupric copper chelates with cystine. Both of these forms are insoluble. Cuprous copper may also reduce cystine to cysteine.

The described reactions can:

• Make cystine and cysteine unavailable for cell use;
• Make a cell culture medium toxic;
• Change the ratios and amounts of cysteine and cystine in liquid media. This is important because cell types vary in their ability to uptake cysteine and cystine.