We have been using DMEM F12 for differentiation of mesenchymal stem cells and we presumed that different large commercial manufacturers stick to the same or very similar composition. Comparing the compositions though we found differences such as Amimed’s DMEM F12 HAM, which (didn't work well for osteogenesis and compared with other DMEM F12 media (LONZA and Sigma). The most notable difference is pyruvic acid sodium salt (55 mg/l Amimed, 110 mg/l Lonza and Sigma) – this can potentially make a big difference. Has anybody paid attention to these differences as well?
Now to complicate the situation further Sigma's powder has 110 mg/l, while liquid formulations are also at 55 mg/l. Gibco' liquid is at 110 mg/l. I will be very happy if somebody can give me an explanation. I don't think it is hydration of the molecule - at least it is not noted.
If you really think it is the pyruvate levels, you could try to supplement the Amimed DMEM/F12 formulation with extra pyruvate and see if this rescues differentiation efficiency.
I have only worked with 110mg/L pyruvate concentrations in my MSC cultures, so do not know whether a 2-fold decrease in pyruvate is pivotal for MSC differentiaition. However, it is conceivable that shifts in pyruvate concentration would alter cellular redox potential and glycolytic rates, so it isn't a stretch to envision that this could matter for osteogenic differentiation.
Hi Jivko,
DMEM is a bit unusual in that it has many formulations which are slightly different. Its probably mutated more that most as it is quite an old formulation. It comes with high (2.5g/l) or low (1g/l) glucose (occasionally none). I assume you are using 1g/l which is the same as F12.
It also comes with varying combinations of pyruvate. The pyruvate is a source of anti-oxidant but it is also an energy source during glycolysis. So its importance increases as anearobic respiration increases. 3D culture, or large media volumes in 2D culture increases anearobic respiration making pyruvate more useful.
Hams F12 has 110 mg/L. If it is mixed with DMEM with high pyruvate (also 100 mg/l) you will get 110 mg/l final. If it is mixed with pyruvate free DMEM you get 55 mg/L average.
I guess it just comes down to which DMEM base the suppliers deem the classical one.
I forgot to mention: If you proliferate the cells in medium with 110mg/l pyruvate, you condition the cells. If you the transfer them to 55mg/l they will have general difficulties irrespective of osteogenesis, they will just be stressed. If you start with 110mg/l stay with 110mg/l for the differentiation.
Pyruvate may play a role in osteogenesis, it would just be a bit difficult to dissect the general stress from the osteogenesis part.
Dear Martin and Daniel, thank you for the detailed answer. I was suspecting that high pyruvate is needed because we expand our cells in hypoxia, hence higher glycolisis rate but didnt think of the pure stress factor. However I have heard of high piruvate being pro-scenescence, so would you think it would be better to start with low pyruvate from the beginning?
No, I haven't seen evidence of pyruvate inducing scenescence, and I think it would be detrimental to cell viability and proliferation if you lowered exogenous pyruvate under low oxygen conditions. What studies/evidence support the pyruvate-scenscence link?
Unfortunately such studies are not published - I was told this by somebody who was developing MSC expansion media for a company. Neither charm nor additional pints of beer got more information, probably he sensed I was too interested.
DMEM12 was always tested in badges taken from different companies for our neuron culture. Once you knew the best badge, we bought in large quantities. Due to the differences of production we always tested in advance. It overcomes a lot of trouble!
Hi Enrico - yes, I agree - we stock too. The only problem is that when we buy - lets say 60 bottles - sometimes they come with a best before date expiring in couple of months and then people don't feel comfortable to use media with a past best before date.
At several companies you could make a reservation for the badges you were testing.
to Saeed:
DMEM/F12 is a mixture of Ham´s F12 media + DMEM. So DMEM/F12 is richer in amino acids, some vitamins and so on. Both can be also mutaded as mentioned Martin J Stoddart (sodium pyruvate and glucose content), so it depends on the variant that you use.
You can check and compare the complete formulation - LifeTechnology has it available for most of their media. e.g. http://www.lifetechnologies.com/cz/en/home/technical-resources/media-formulation.216.html
Hope that will help.
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