Amino acids Amino acids are the basic units that make up proteins. Different types of cells have different requirements for amino acids, but there are several types of amino acids that cells cannot synthesize themselves and must rely on the culture medium. These types of amino acids are called essential amino acids. Among them, glutamine is Cells synthesize essential amino acids for nucleic acids and proteins. In the absence of glutamine, cells grow poorly and die. Therefore, there are large amounts of glutamine in various culture solutions. However, glutamine is very unstable in solution It should be stored in a refrigerator at -20 ℃ and added to the culture solution before use. When the culture solution containing glutamine has been stored in the refrigerator at 4 ℃ for more than 2 weeks, the original amount of glutamine should be added again.
Carbohydrates Carbohydrates are the main energy source for cell growth, some of which are components of synthetic proteins and nucleic acids. Mainly are glucose, ribose, deoxyribose, sodium pyruvate and acetic acid.
The main function of the inorganic salt in the inorganic salt culture solution is to help the cell maintain the osmotic pressure balance. In addition, by providing sodium, potassium and calcium ions, it helps the cell to regulate the function of the cell membrane. The osmotic pressure of the culture solution is a very important factor. Tolerates 260mOsm / kg 320 mOsm / kg. The osmotic pressure of the standard culture fluid fluctuates within this range. Special attention: adding other substances to the culture fluid may significantly change the osmotic pressure of the culture fluid, especially soluble in strong acids or strong Substances in alkali. When adding HEPES to the culture broth, adjust the sodium ion concentration as follows.
The pH required for most cells in the buffer system is 7.2-7.4. However, the optimal pH for cell culture varies with the type of cell cultured. Fibroblasts prefer a higher pH (7.4-7.7), which is required for the passage of transformed cell lines Partially acidic pH (7.0-7.4). Since most culture fluids are buffered by sodium bicarbonate (NaHCO3) and CO2 systems, the CO2 concentration in the gas phase should be balanced with the sodium bicarbonate concentration in the culture fluid. If the gas phase or incubator The concentration of CO2 in the air is set at 5%, and the amount of NaHCO3 added to the culture is 1.97g / L; if the CO2 concentration is maintained at 10%, the amount of NaHCO3 added to the culture is 3.95g / L. The cell culture bottle cap should not be Tighten too tightly to ensure gas exchange. HEPES is a non-ionic buffer that has good buffering capacity in the pH range of 7.2-7.4, but is very expensive 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 HEPES. Under this culture condition, the cap of the cell culture flask should be tightened to prevent the required A small amount of carbonate is scattered into the air. Most of the culture fluid contains phenol red pH indicator, orange yellow acidic medium, the alkaline broth was dark red.
Vitamins In cell culture, although serum is an important source of vitamins, many mediums are supplemented with various vitamins to suit the growth of more cell lines.
Other components include some other components in some more complex culture fluids. For example, the DMEM culture fluid commonly used in hybridoma technology needs to be supplemented with sodium pyruvate and 2-mercaptoethanol (2-Mercaptoethanol, 2-Me ). 2-Me has a very important effect on cell growth. Some people think that it is equivalent to fetal bovine serum, which directly stimulates cell proliferation. The active part of 2-Me is the sulfhydryl group, one of the important functions is to make Sulfur compounds are reduced to glutathione, which can induce cell proliferation, which is a non-specific activation. At the same time, it avoids the damage of peroxide to cultured cells. Another important role is to promote mitogen response and DNA synthesis, increase plants The effect of lectin (PHA) on the transformation of lymphocytes has been widely used in hybridoma technology. In addition, it has also been used in some cells that are difficult to culture. 2-Me is a small molecule reducing agent that is very easy to oxidize. The molecular weight is 78.13 , Pure 2-Me is a colorless and pungent liquid, specific gravity is 1.110-1.120 (Do20), commonly used final concentration is 5 × 10-5M. It is often formulated into 0.1M storage solution, used per liter of culture solution Add 0.5ml.
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