Cell culture medium is the nutrient basis for artificially imitating the in vivo growth environment of animal cells and maintaining the survival and proliferation of cells in vitro. a nutrient. So what are the common problems encountered in the use of cell culture medium? Let's do an analysis.
1. Buffer system selection and pH change of cell culture medium
Since the optimum pH for most cells is between 7.0 and 7.4, deviations from this range may have deleterious effects on cell growth. However, the pH requirements of various cells are not exactly the same. Primary cultured cells generally have poor tolerance to pH fluctuations, while infinite cell lines have strong tolerance. Therefore, in the primary culture, the buffer system in the culture medium is more important. The general cell culture medium uses a balanced salt system, but different culture media or the same series of culture media use different balanced salt systems. For example, 199 series and MEM series all have Hanks' system culture medium and Earle's system culture. base. Some media are not the above-mentioned conventional balanced salt system, such as RPMI1640 medium, F12 medium. The balanced salt system of the MEM low serum medium is also not a conventional balanced salt system, and the buffering capacity of the balanced salt system is stronger than that of the conventional balanced salt system.
cell culture
There are many reasons for pH drop during cell culture. When the cells are growing very fast, the pH value usually drops rapidly, which can be solved by timely passage, increasing the passage ratio, or reducing the serum volume. In addition, overtightening of the culture bottle cap, insufficient buffering capacity of the NaHCO3 buffer system, incorrect salt concentration in the culture medium, bacterial, yeast or fungal contamination can also cause the pH to drop often rapidly. At this time, you can solve it in the following ways:
1) Increase the NaHCO3 concentration in the culture medium or decrease the CO2 concentration in the incubator. When the NaHCO3 content is between 2.0 g/L and 3.7 g/L, the corresponding CO2 concentration is 5~10%;
2) Switch to a CO2-independent culture medium;
3) Properly loosen the bottle cap. Add HEPES buffer to the culture medium to make the final concentration 10-25 mM;
4) Use the culture solution based on Earle's salt in the CO2 culture environment, and use the culture solution prepared by Hanks' salt in the atmospheric culture environment;
5) Discard culture or sterilize with antibiotics if contamination is the cause.
2. Several important additives are commonly used in cell culture media and problems that should be paid attention to in the use process
Phenol red is used as an indicator of pH in cell culture media. In general, the pH value of the medium can be judged by the indicator effect of phenol red, but the content of phenol red in low serum or serum-free cell culture medium is different from that in ordinary cell culture medium, and cannot be observed by naked eyes or The pH value is determined empirically, and it is recommended to use a pH meter for measurement. Phenol red usually does not have a significant effect on the quality of biological products produced in serum-containing cell culture media, and can also be removed by purification techniques, but phenol red may cause intracellular sodium/potassium imbalance in serum-free cell culture media, affecting cell growth.
cell culture plate
Sodium bicarbonate is mainly used as a buffer system in cell culture medium, and also has the effect of regulating osmotic pressure. Usually the recommended amount of sodium bicarbonate in the product instructions for use is a standard and safe amount, which is obtained based on practical experience on a scientific basis. However, due to different cell lines (strains), the same cell may adapt to different environments (different cell tolerance, etc.), and there are regional differences in water quality, etc., which can also be slightly modified in the actual production process, but the use of Those who need to do the corresponding tests (physicochemical and cell production tests, etc.).
HEPES is a non-ionic buffer with good buffering capacity in the pH range of 7.2 to 7.4, and may be toxic to some cells at high concentrations. HEPES buffer can be used with low levels of sodium carbonate (0.34g/L) to offset the increase in osmotic pressure caused by the addition of additional HEPES. Its safe concentration range is 10 ~ 25mmol/L.
Sodium pyruvate can be used as an alternative carbon source in cell culture, and although cells prefer glucose as a carbon source, cells can also metabolize sodium pyruvate in the absence of glucose.
Glutamine is very unstable in solution. It can be decomposed to 50% at 4 ℃ for 1 week. It is best to prepare it separately in use, store it in a -20 ℃ refrigerator, and add it to the cell culture medium before use.
