Glutamine is a non-essential amino acid for the human body. However, mammalian cells in culture require millimolar concentrations of this amino acid, which is essential for cell growth and proliferation, particularly in rapidly dividing cells such as cancer cells.
Glutamine participates in numerous intracellular metabolic reactions, acting as both a nitrogen and carbon donor (Zhang et al., 2017).
👉 For this reason, all cell culture media must contain glutamine.
Glutamine concentration
The concentration of glutamine required in culture media varies depending on the cell line.
In most cases, a concentration of 2 mM is commonly used.
Glutamine in commercial media
When possible, it is preferable to avoid purchasing media already supplemented with free glutamine.
Alternatively, media containing glutamine in stable dipeptide forms, such as:
- alanyl-L-glutamine
- glycyl-L-glutamine
are often preferred.
In fact, glutamine is unstable in aqueous solution and degrades non-enzymatically over time.
This degradation leads to the formation of pyrrolidonecarboxylic acid and ammonia, both toxic to cells.
Because of this instability glutamine is typically added to the medium just before use. After supplementation, the medium can be stored at 4°C for up to 2–3 weeks.
Commercial media pre-supplemented with glutamine may already have lower-than-nominal glutamine concentration and increased ammonia levels. Indeed, at physiological pH, ammonia is mainly present as the NH₄⁺ ion. In vivo, ammonia is converted to urea and eliminated. In cell culture, however, ammonia accumulates in the medium, leading to toxic effects on cells.
These effects occur both at 20°C and at 4°C (Heeneman et al., 1993).
Serum concentration has little or no effect on glutamine degradation rate (Ozturk et al., 1990).
Preparation of a 200 mM glutamine stock solution.
Although commercial solutions are available, a glutamine stock solution can be easily prepared in the laboratory at lower cost.
Protocol
- Weigh 2.92 g of L-glutamine powder
- Dissolve it in approximately 90 mL of PBS
- Stir using a magnetic stirrer until completely dissolved
- Adjust the final volume to 100 mL with PBS
- Sterilize the solution by filtration using a 0.22 µm filter
⚠️ Do NOT autoclave
High temperature causes glutamine degradation.
-
Aliquot the solution into small volumes
👉 this avoids repeated freeze–thaw cycles -
Store at −20°C
👉 The solution is stable for up to 6 months