L-Glutamine in Cell Culture

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

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

L-glutamine is an unstable essential amino acid required in cell culture media formulations. Most commercially available media are formulated with free L-glutamine which is either included in the basal formula or added to liquid formulations at time of use. L-glutamine is unstable at physiological pH in liquid media. It breaks down to ammonium and pyroglutamate at rates that make it a problem in many biomanufacturing applications. Today several proprietary media used in biomanufacturing are supplemented with L-glutamine in dipeptide forms, such as alanyl-l-glutamine and glycyl-l-glutamine. A less well defined source of L-glutamine comes from the use of protein hydrolysates, especially gluten hydrolysates.

The concentration of L-glutamine used in classical media ranges from 0.5 mM in Ames' Medium to 10 mM in MCDB Media 131. The more typical concentrations in media used for biomanufacturing and tissue engineering application is between 2 and 4 mM. DMEM/Ham's Nutrient Mixture F-12 (50:50) is often used as a starting formulation for proprietary media used with Chinese Hamster Ovay, CHO, cells. L-glutamine in DMEM/F12 Nutrient Mixture is 2.5 mM. L-glutamine in Serum-Free/Protein Free Hybridoma Medium is 2.7 mM. L-glutamine in DMEM, GMEM, IMDM and H-Y medium is 4 mM. IMDM is often used as a starting formulation for proprietary hybridoma cell culture media. Hybridoma cells grow better in concentrations of L-glutamine that are above the average levels found in media.

Because of its chemical instability and importance for cell growth and function, it is critical that the delivery of L-glutamine be optimized to each unique cell culture process. Hence the effective use of L-glutamine and L-glutamine equivalents in cell culture requires an understanding of its chemistry and multiple delivery forms. For a more complete discussion of L-glutamine as a cell culture additive go to Sigma's Media Expert.

Primary Functions of Glutamine in Cell Culture Systems:

Glutamine supports the growth of cells that have high energy demands and synthesize large amounts of proteins and nucleic acids. It is an alternative energy source for rapidly dividing cells and cells that use glucose inefficiently. Cells require nitrogen atoms to build molecules such as nucleotides, amino acids, amino-sugars and vitamins. Ammonium is an inorganic source of nitrogen that exists primarily as a positively charged cation, NH4+, at physiological pH. Ammonium nitrogen used by cells is initially incorporated into organic nitrogen as an amine of glutamate or an amide of glutamine. These two amino acids provide the primary reservoirs of nitrogen for the synthesis of proteins, nucleic acids and other nitrogenous compounds.

Reactions that fix nitrogen into glutamate and glutamine consume energy equivalents. Glutamate is synthesized from ammonium and alpha ketoglutaric acid, a tricarboxylic acid (TCA) cycle intermediate. Its synthesis requires the oxidation of either NADH or NADPH. Glutamine is formed from ammonium and glutamate and its synthesis consumes ATP. The enzymes involved in glutamate synthesis, glutamate dehydrogenase (EC and glutamate synthase (EC are reversible. The enzyme responsible for glutamine synthesis, glutamine synthetase (EC, is highly regulated to limit the production of glutamine to cell requirements. The catabolism of glutamine to glutamate and ammonium is mediated by mitochodrial enzymes called glutaminases (EC ). Ammonium produced in vivo can be metabolized to urea. In vitro, ammonium is not metabolized to urea. Under some in vitro conditions, ammonia accumulates in the extracellular medium as ammonium ion.

Roles of glutamine:

  • Glutamine contains one atom of nitrogen as an amide and another atom of nitrogen as an amine and it transports and delivers nitrogen to cells in quantities that are toxic as free ammonium.
  • Glutamine amide nitrogen is used in the synthesis of the vitamins NAD and NADP, purine nucleotides, CTP from UTP and asparagine. Nitrogen initially stored in glutamine can also be used to produce carbamyl phosphate for the synthesis of pyrimidines.
  • Glutamine is a precursor of glutamate, a key amino acid used for the transamination of alpha ketoacids to form other alpha amino acids.
  • When glucose levels are low and energy demands are high, cells can metabolize amino acids for energy. Glutamine is one of the most readily available amino acids for use as an energy source and it is a major source of energy for many rapidly dividing cell types in vitro.

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


Molecular formula: C5H10N2O3

Molecular weight: 146.15

Isoelectric point (pH): 5.65

Pka = 2.17 and 9.13

Glutamine Stability:

L-Glutamine is a freely soluble neutral amino acid containing an R-group amide. It can break down non-enzymatically into ammonia and pyroglutamate (pyrrolidonecarboxylic acid) in liquid media. The breakdown of L-glutamine over time is dependent on pH, temperature and the presence of various anions. Glutamine deamination occurs in both acidic and basic conditions and it is significantly more rapid in the presence of phosphate or bicarbonate. The deamination rate at a fixed phosphate concentration increases as the pH increases from 4.3 to 10. The rate of deamination in the presence of phosphate increases almost linearly as the pH increases from 7 to 8.

When glutamine is present as an amino acid residue in proteins or peptides, it is stable. Glutamine is available for cell culture as a dipeptide or a protein hydrolysate. Wheat gluten is a rich source of peptidyl glutamine that has been used successfully to culture recombinant Chinese Hamster Ovary cells (rCHO) in serum-free, animal-protein-free media.

Glutamine 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.