The cell culture medium must contain sufficient nutrients to meet the substances and energy required for biochemical reactions such as new cell synthesis and cell metabolism. The main components of cell culture medium are water, amino acids, vitamins, carbohydrates, inorganic salts and other auxiliary nutrients. Therefore, we must pay attention to the sterilization method of cell culture medium and related precautions in daily experiments.
cell culture
1. Sterilization methods and precautions for different cell culture media
Sterilization methods of cell culture medium are divided into autoclave sterilization and membrane filtration sterilization. Different culture mediums may have different sterilization methods due to their different nutrients.
Autoclave
Some media, such as MEM, can be autoclaved, these media generally do not contain L-glutamine and sodium bicarbonate, and are generally added after the media has been autoclaved. In addition, high-pressure glutamate (such as L-alanyl-L-glutamine) can be used instead of L-glutamine. The autoclavable medium is fully sterilized with minimal loss of nutrients at 121°C, 15 psi, and 15 minutes, without extending the sterilization time.
Most cell culture media are not suitable for autoclaving. Because the culture medium often contains vitamins, proteins, polypeptides, growth factors and other substances, these substances are prone to denaturation or loss of function under high temperature or radiation exposure, so the above-mentioned liquids are mostly filtered and sterilized to remove bacteria. There are many filter membranes available for filtration and sterilization, and their materials are mostly PES, nylon, polycarbonate, cellulose acetate, nitrocellulose, PTFE, ceramics, etc. Membrane filtration sterilization is a commonly used and convenient method at present. Filter membranes with 0.2 μm pore size are often used, and some use 0.1 μm pore size. Compared with high pressure filtration, membranes have a lifespan and are more expensive, but are less destructive to the nutrients in the cell culture medium.
2. Precautions during storage of different cell culture media
Generally, liquid cell culture medium should not be frozen at -20 ℃, because nutrients may be precipitated during thawing, which will affect the culture effect. Under normal circumstances, it should be stored at 2 ~ 8 ℃ away from light. Before use, take it out of the refrigerator and put it at room temperature for equilibration. The usual liquid medium is valid for 6 to 12 months. Try to avoid long-term storage of liquid cell culture medium. The glutamine in it will decompose slowly with the extension of storage time. If the cell growth is not good, you can consider testing the glutamine content in the medium to determine whether to add glutamine. . Commercially available liquid cell culture medium has a specific expiration date. After using dry powder cell culture medium to prepare liquid, it should also be stored at low temperature (2 ~ 8 ℃). In addition to the easy degradation of glutamine in the medium, other components in the medium may also be degraded or precipitated with increasing temperature.
