Cell culture is the most common and basic experiment in the laboratory, but it is not the simplest. Don't underestimate cell culture, there is a big problem here. Sometimes the cell state is not good, and it is impossible to do experiments such as transfection and drug screening. There are many factors that affect cell culture. Let's talk about culture medium and serum first.
◆ MEM is developed from Eagle's basic medium (BME), in which the range and concentration of components are increased.
The improved BEM(DMEM) medium is designed for mouse fibroblasts, and now it is often used to cultivate adherent cells. The amino acid concentration of DMEM is twice that of MEM, and the vitamin concentration is four times that of MEM. The double concentration of HCO3- and CO2 played a better buffering role. In the original formula, the glucose content was1000 mg/L. Later, for the growth of some cells, the glucose content was adjusted to 4500 mg/L. This is what people often say is low sugar and high sugar.
◆ aMEM contains extra amino acids, vitamins, nucleosides and fatty acids, which can be widely used in various cell types, including cells with strict requirements on nutritional components.
Ham's F 12 was designed to clone CHO cells at low serum concentration, and now it is widely used in the analysis of clone formation rate and primary culture. F 12 can also be mixed with DMEM in equal volume to obtain products with high concentration and diverse components. This culture medium has been used in many primary cultures and cell lines that are more difficult to cultivate.
◆ RPMI 1640 medium is specially designed for lymphocyte culture and has been widely used in suspension cell culture.
Prepare several kinds of culture media, and then spend about two weeks doing a simple cell growth experiment to select the most suitable one.
In the past, most laboratories used dry powder culture medium, but the preparation process was complicated, there might be some concentration errors in the process of dissolution, pH adjustment and filtration, and the water quality in some laboratories was not ideal, so the culture effect would be different.
Selection of serum:
Serum containing growth factor can promote cell proliferation, and serum containing adhesion factor can promote cell adhesion, and it also has antitrypsin activity. Serum is also a source of minerals, lipids and hormones. The most commonly used sera are calf serum, newborn bovine serum, fetal bovine serum and horse serum. Bovine serum is classified according to the time of blood collection. The earlier the blood collection time, the richer the nutrition and the less antibodies, so fetal bovine serum is suitable for demanding cell lines and clone culture.
The difference between serum batches is inevitable, which comes from different preparation methods and sterilization methods, the age difference of animals and the storage conditions of serum, and is closely related to the source of blood collection animals. Different pastures, different climates and other environmental factors may affect the quality of serum. Therefore, if you choose a serum, you should use it as long as possible. When it needs to be replaced, it should be as similar as possible to the original batch of serum. Many companies now provide serum reserves. Once a certain batch of serum is selected, it is best to reserve it for one year, which can avoid a lot of trouble.
Serum is an indispensable component in cell culture, but different batches of serum vary greatly. When replacing serum, a lot of tests are needed to ensure that the replaced serum is similar to the original serum. The supply of serum is also a factor that must be considered.
Serum-free medium, usually expressed as SFM, as the name implies, means that serum is not needed to be added in cell culture, but growth factors or cytokines may be added in some applications. Adding the main components of serum, such as adhesion factors, growth factors, essential nutrients, hormones, etc., to serum-free medium can reduce the unfavorable factors brought by the above serum and make the cell culture conditions more stable. However, it is not perfect, and the conditions for the transition from serum-containing culture to serum-free culture are not as direct as expected. Cells at different stages of differentiation and development (for example, compared with directional precursor cells) need different formulations, and the selection of growth factors and cytokines is particularly important. Moreover, while removing serum, it also removes the protection and detoxification of some serum proteins, so the purity of reagents and water and the cleanliness of instruments are required to be higher. In addition, its price is much more expensive than ordinary culture medium.
So many kinds, it is also a headache to choose. Except for some publicly prepared media, such as MCDB 13 1 (Sigma) for culturing endothelial cells, most liquid media are patented, that is, the ingredients in them are confidential. Then you can only search the literature, find a supplier to recommend, and then do several subcultures with your own cells to select the most suitable medium. After all, practice makes true knowledge.