Essential and Non-Essential Amino Acids in Cell Culture

The terminology is borrowed from nutrition but the list is not the same, and this is the source of most confusion around the question.

In cell culture, an amino acid is operationally essential if the cells in your flask cannot synthesise it in sufficient quantity, so it has to be present in the medium or growth stops. Harry Eagle established this list experimentally in the 1950s by removing components one at a time and observing which omissions cells could not survive.

The cell-culture essential set is thirteen amino acids: L-arginine, L-cystine, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

Four of those - arginine, cystine, tyrosine and glutamine - are not classed as dietary essentials, because a whole organism has tissues and pathways that a monolayer in a flask does not. Cultured cells lack the interorgan metabolism that supplies them, so from the medium's point of view they must be provided. It is worth being precise about this when reading older literature: a paper describing tyrosine as non-essential is almost certainly using the nutritional definition, not the culture one.

A commercial MEM Essential Amino Acids 50X supplement contains twelve of the thirteen. Glutamine is left out and supplied separately, for reasons covered below.

The non-essential set used in cell culture is exactly seven: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine. These are the contents of every product sold as MEM Non-Essential Amino Acids.

The standard 100X formulation contains all seven at 10 mM each - glycine 750 mg/L, L-alanine 890, L-asparagine 1320, L-aspartic acid 1330, L-glutamic acid 1470, L-proline 1150 and L-serine 1050. Diluted 1:100 by adding 10 mL per litre of medium, each ends up at 0.1 mM final concentration. That the concentrate is a clean 10 mM across all seven is deliberate and makes the arithmetic trivial: 1X always means 0.1 mM of each.

Note what is not in it. There is no glutamine in a NEAA supplement, and no cysteine. If a protocol says to add NEAA and glutamine, those are two separate additions, and adding one does not cover the other.

This is the reasonable objection, and there are four answers.

Biosynthesis is not free. Making an amino acid from scratch consumes carbon skeletons drawn out of central metabolism, ATP, and reducing equivalents. In a rapidly proliferating culture, those are the same resources being used to build biomass. Supplying the amino acid preformed lets cells spend that budget on growing instead, and the usual result is a modestly higher growth rate and a higher achievable density.

Some lines cannot actually make them. "Non-essential" describes the typical mammalian cell, not every cell line. CHO-K1 is proline-auxotrophic through a deficiency in pyrroline-5-carboxylate synthase and simply requires proline in the medium; expressing the missing enzyme restores proline independence. This is not a curiosity - it is why Ham's F-12, the medium developed for CHO cells, carries proline at 0.3 mM, three times the concentration of the other six. Similarly, lines with low asparagine synthetase activity depend on exogenous asparagine.

Some of them do more than build protein. Serine and glycine feed one-carbon metabolism through the folate cycle, supplying units for nucleotide synthesis and methylation reactions. A proliferating cell's demand for serine can exceed what it conveniently synthesises, and the two are interconvertible, so the pair is often limiting together.

Uptake is not guaranteed by presence. Aspartate in particular is transported poorly by many mammalian cells, so its concentration in the medium is a weak predictor of its availability inside the cell. This is worth remembering before concluding that a medium containing aspartate has covered that requirement.

Against all this, the supplement is inexpensive and adds about 0.7 mOsm/kg to the medium, which is negligible against a typical 280-320 mOsm/kg. The cost of adding it when it was not needed is low; the cost of omitting it when it was needed is a slow culture you spend a fortnight troubleshooting.

This is the practical question, and the answer varies far more than most people expect. The comparison table below gives the full breakdown; the summary is:

MEM (Eagle's) contains none of the seven. This is by design - it is the minimum essential medium, and the NEAA supplement exists precisely to complement it. Note that a bottle labelled EMEM from ATCC does include non-essential amino acids, while a Gibco MEM does not, so "MEM" alone does not tell you.

DMEM contains two: glycine and L-serine, both at 0.4 mM. It carries none of the other five. DMEM is therefore a common candidate for NEAA supplementation, particularly for HEK293 work, transfection and virus production.

RPMI 1640 contains six of the seven, at concentrations that differ from each other - asparagine 0.379 mM, serine 0.286, proline 0.174, aspartic acid 0.150, glutamic acid 0.136 and glycine 0.133. It contains no L-alanine. If you are supplementing RPMI, alanine is the only member of the set genuinely missing.

Ham's F-12 contains all seven, six at exactly 0.1 mM and proline at 0.3 mM - the same working level a 1X NEAA addition would give. Supplementing F-12 with NEAA is normally redundant.

IMDM contains all seven, and generally at higher levels than F-12: glutamic acid 0.510 mM, glycine and serine 0.4, proline 0.348, alanine 0.281, aspartic acid 0.226 and asparagine 0.189.

DMEM/F-12 contains all seven, at roughly half the F-12 concentrations because it is a 1:1 blend with DMEM.

MEM alpha contains them, as part of its enrichment over plain MEM.

Glutamine sits in the essential list but behaves unlike anything else in it, and it is the amino acid most likely to cause trouble.

Cells use it as the principal nitrogen donor for biosynthesis and, through glutaminolysis, as a major carbon source feeding the TCA cycle. Demand is high - MEM carries 2 mM and DMEM 4 mM, an order of magnitude above most other amino acids.

It is also chemically unstable in aqueous solution. Free L-glutamine cyclises spontaneously to pyrrolidone carboxylic acid, releasing ammonia, and the reaction runs faster at 37 C than in the cold. Two problems follow: the medium loses glutamine over its shelf life and during incubation, and the ammonia that accumulates is itself inhibitory to many cell lines.

Three responses are standard. Buy medium without glutamine and add it fresh from a frozen 100X or 200X stock, which is the traditional approach. Use a dipeptide form - L-alanyl-L-glutamine or L-glycyl-L-glutamine - which is stable in solution and is cleaved by cell-surface peptidases to release glutamine as it is needed, avoiding both the loss and the ammonia spike. Or buy a medium formulated with the dipeptide already in it, which several products in most catalogues are.

The practical point for this page is simply that glutamine is never covered by a NEAA supplement and rarely covered adequately by an old bottle of complete medium. If a culture is growing poorly in medium that has been on the shelf for months, glutamine is among the first things to check.

Adding NEAA. Add 10 mL of a 100X supplement per litre of medium, giving 0.1 mM of each of the seven. Add it to the complete medium once, at the time you supplement with serum and glutamine, and record it as part of the formulation rather than as an occasional extra - inconsistent supplementation between batches is a common and completely avoidable source of variability.

Media that usually benefit: MEM and DMEM, which contain none or only two of the seven. NEAA supplementation of DMEM is routine for human pluripotent stem cell culture, HEK293-based transfection and virus production, hybridoma work, and mesenchymal stromal cells.

Media where it is usually redundant: Ham's F-12, IMDM and DMEM/F-12 already carry all seven at working concentrations. Adding more is not harmful at these levels but it is not doing anything either, and it makes your formulation harder to reproduce.

RPMI 1640 is the interesting middle case - it has six of the seven and lacks only alanine, so a full NEAA addition mostly tops up what is already there.

Do not treat NEAA as a rescue for a failing culture. If cells are growing badly, contamination, glutamine degradation, serum lot, pH and mycoplasma are all more likely explanations, and adding amino acids to an already-adequate medium will not help. Change one variable at a time.

Do not confuse the two supplements. MEM Non-Essential Amino Acids 100X supplies the seven at 0.1 mM. MEM Essential Amino Acids 50X supplies the twelve essential amino acids other than glutamine, and exists to fortify or reconstitute media rather than to be added routinely to a complete formulation. They are not interchangeable and the concentrates are at different strengths.