I have a few questions about the preparation of cell culture medium, and I'd like to clarify some practices that I'm not entirely sure about. First, is there a specific relationship between the amount of sodium bicarbonate and HEPES added when making the medium? Does my current method affect the quality of the medium, and how significant is this impact? Also, can I add 3.7g/L of carbonic acid as instructed in DMEM? In addition to adding 20mM HEPES, what else should I consider?
When I looked up other literature, I had some doubts. Some papers mention using Hyclone DMEM and only adding 1.97g/L of sodium bicarbonate along with 20mM HEPES. After preparation, they adjust the pH to 7.2–7.4 using 10M NaOH, ensuring it doesn't exceed 7.5 after filtration. However, when I tried preparing the medium myself, I used 1M NaOH to adjust the pH, but the required amount was quite large. So I switched to using 10M NaOH as suggested in the literature, and followed the DMEM instructions to add 3.7g/L of sodium bicarbonate.
Another thing I'm concerned about is how I add serum during the preparation. I didn’t account for isotonicity between the serum and the medium. For example, I calculated 5% serum volume by dissolving the DMEM dry powder in 950mL of water, then added 50mL of serum. Is this approach correct?
If the medium I prepared this way has a major impact on cell culture, is there a way to fix the existing 500mL of medium, and if so, how should I do it?
Also, I’m worried about overusing glutamine. Our lab’s dry powder media aren’t always made in one full 1L package; sometimes we make 200mL or 400mL portions. The remaining powder is stored at 4°C, but occasionally it's not sealed tightly. When reused, the powder might become moist or clump, which could affect nutrient stability—especially glutamine, which may degrade. If I use clumped dry powder, I want to re-add glutamine, but I’m afraid of overdoing it. Any advice on this?
To summarize:
1. There is no strict requirement between the amounts of sodium bicarbonate and HEPES. Sodium bicarbonate forms a carbonate buffer system with CO₂ in the incubator, while HEPES is an organic buffer with higher buffering capacity. Sodium bicarbonate should be added according to the standard dosage, and HEPES can be added at 10–25 mM as an auxiliary buffer.
2. When preparing the medium, whether to include serum volume depends on the protocol. Some studies suggest considering serum volume to maintain nutrient concentration, but most modern methods don’t factor it in, and the medium is usually prepared for 1L.
3. Dry powder should be dissolved in one go because mixing isn’t uniform. Glutamine solutions are unstable, so it’s better to prepare fresh solutions rather than re-add them later.
Additionally, the medium doesn’t need pH adjustment after dissolution, as it’s formulated to pH 7.4. Adding alkali can lead to CO₂ loss, affecting the buffer system.
Regarding my training program, I’ve read that too much serum can promote the growth of unwanted cells. Many cell lines are now successfully cultured in serum-free media. However, serum-free cultures require specialized media with specific cytokines, which is different from simply omitting serum.
I’d really appreciate any insights or suggestions you might have.
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