Do DMEM low sugar MSCs require additional supplementation of sodium pyruvate and glutamine?

Supplementing with sodium pyruvate and glutamine generally does not directly damage primary cells due to excessive supplementation, provided that the final concentration does not exceed the literature range too much, especially when using 1% sodium pyruvate and mistakenly adding 100 mM mother liquor to 10%. What is even more concerning is the storage time of the mother liquor: Glutamine is prone to degradation and produces ammonia at 4 ° C and 37 ° C, which can affect MSCs. In the original system construction stage, the fewer variables, the better, so if you can buy pre mixed DMEM low sugar, buy pre mixed, which is more stable than manual replenishment. If you are concerned about the impact, you can set up one group without supplementation and one group with supplementation to compare the adhesion rate and early proliferation.

After opening a 500 mL bottle, it is recommended to aliquot immediately, but DMEM liquid is not recommended for repeated freeze-thaw cycles. A common practice is to aseptically dispense into 50 mL centrifuge tubes, keep a small portion at 37°C or sample it for sterility testing, and store the rest at 4°C protected from light, using it within 4-6 weeks if possible. If freezing at -20°C is absolutely necessary, phosphate precipitates may form after thawing; gently swirl at room temperature to check whether they redissolve, and the CO2 equilibrium must be re-established before measuring pH and observing color and clarity. During the MSC line establishment stage, it is recommended to prioritize ensuring fresh culture medium and not rely on frozen stock solutions.

It is recommended to directly purchase DMEM low sugar products containing GlutaMAX and sodium pyruvate. GlutaMAX is more stable than regular L-glutamine, and primary cell culture is less likely to produce ammonia due to glutamine degradation. Supplementing with 1% sodium pyruvate and 2 mM glutamine is a common final concentration in literature, and self mixing can easily introduce variables due to repeated freezing and thawing or filtration of the mother liquor. The finished product eliminates the need for weighing, pH adjustment, and filtration, and also reduces the risk of contamination. Before purchasing, ask the agent to provide a COA to confirm if it contains phenol red, NaHCO3, and sodium pyruvate. If the budget is not particularly tight, try to use finished products during the original construction phase to ensure more consistent conditions.

If you have already purchased regular low-glucose DMEM and need to supplement it: add 100 mM sodium pyruvate stock solution at 1:100 to a final concentration of 1 mM; add 200 mM L-glutamine at 1:100 to a final concentration of 2 mM. The glutamine stock solution must be aliquoted and stored at -20°C, avoiding repeated freeze-thaw cycles and prolonged storage at 37°C. After supplementation, mix well in a biosafety cabinet, preferably filter through 0.22 µm and then aliquot. FBS itself contains some nutrients, but primary MSCs have a relatively high demand for glutamine, so it is recommended not to omit it. First prepare a small test batch and observe primary cell attachment before using it for full-scale line establishment.