1. All equipment, devices and solutions in contact with cells must be kept absolutely sterile to avoid being contaminated by extracellular microorganisms.
At present, the most reliable and commonly used sterilization method is high pressure steam sterilization.
Such as: glass products, cloth products and metal products: 6.8kg for 20min.
Rubber products: 3.6 ~ 4.5 kg 10 min.
Culture medium, such as Hanks solution, hydrolyzed milk protein: 3.6 ~ 4.5 kg 10 min.
Infected articles and animal carcasses in the experiment: 20min 6.8kg.
Test tubes, straws, etc. Can be sterilized by dry heat;
All liquids that are not suitable for heating sterilization, such as synthetic culture solution, calf serum, trypsin and other solutions, can be filtered and sterilized.
Concentration and application of disinfectants commonly used in cell culture
2. There must be sufficient nutrition supply, and there must be absolutely no harmful substances, including tiny amounts of harmful ions. Therefore, attention must be paid to the cleanliness of equipment and the quality of water used for liquid preparation.
Cell culture requires high quality water (see water treatment).
3. Ensure proper oxygen supply.
Cell metabolism needs air, otherwise cells will die. When static culture is carried out in a culture bottle or rotating culture is carried out in a rotating bottle, as long as the amount of culture solution does not exceed 30% of the total capacity, cells can be ensured to have enough oxygen by exchanging with air at the liquid level. Ventilation is necessary when submerged culture is carried out in a tank. Usually, air containing 5% CO2 is used, or CO2, N2, O2 and air are mixed in different proportions as required. Too much oxygen is not conducive to cell growth.
4. Harmful products produced in cell metabolism should be removed at any time.
In the process of metabolism, cells will produce a lot of metabolic wastes, such as CO2, lactic acid, ammonia, urea, indole and so on. These products are harmful to the survival of cells and must be removed in time. In a small amount of culture, when the phenol red indicator in the culture medium is yellow, it means that a considerable amount of lactic acid has been produced, and it is necessary to replace the fresh culture medium in time. In large-scale culture, it is necessary to determine the contents of glucose and lactic acid at any time and adjust the perfusion speed to keep the metabolites at a harmless level.
5. Have a good external environment suitable for its survival, including temperature, pH, osmotic pressure and ion concentration.
Different cells have different requirements for pH. Generally speaking, the pH suitable for cell growth is 6.8 ~ 7.4. Too high or too low is not conducive to cell growth. As mentioned above, cells will produce a lot of lactic acid during metabolism, which will reduce the pH value of the culture. In order to maintain the pH value of the culture medium, various buffer systems are often added to the culture medium, such as Na2HPO4/Na2HPO4, NaHCO3/H2CO3(CO2), triglycine and Hepes solution (commonly used concentration is 10 ~ 50mol/L).
6. Inoculate in time to maintain proper cell density (see cell passage).
Second, cell separation.
1. Isolation of human leukocytes.
Commonly used to prepare IFN-α and γ.
Mainly from the venous blood of blood donors, but also from the residual blood during cardiopulmonary bypass, umbilical cord blood, spleen and tonsils during childbirth.
2. Isolation of human embryonic myometrial cells. Is used for preparing interferon-beta.
3. Calculation of cell age in vitro
The age of diploid cells is calculated by doubling the cell population, based on the number of cells in each culture container cell population, and each doubling is regarded as a generation, that is, 1 bottle of cells is transferred to 2 bottles (1: 2 seed rate) and then the full bottle is regarded as a generation; 1 bottle to 4 bottles (1: 4) is the second generation; 1 bottle and 8 bottles (1: 8) are three generations. The age of producing cells is limited to the first 2/3 of the cell life.
The passage cell line was passaged at a certain dilution multiple, and each passage was passed on to the next generation. According to the actual experience data of each cell line, ensure the quality and safety of cells, and determine the last restricted generation cells for production and use.
Four. Cell recognition test
Newly established cell lines or strains, cell banks and cells at the end of production should be identified as the cells. Genetic characteristics, DNA fingerprints, chromosome markers, immunology, HLA and biochemical methods (such as isoenzymes) can be used to judge, and any one of them can be selected. Simple and identifiable methods can also be used, but they need to be approved by the state drug inspection agency.
Human diploid cell lines are derived from normal human fetal lungs or other tissues, and are mainly used for virus culture and vaccine preparation.
1. cell line requirements
Human diploid cell lines should be comprehensively tested according to the following requirements.
The establishment of (1) cell line must have the following information.
The gestational age and sex of the fetus used to establish the cell line, and the reasons for termination of pregnancy.
The age, occupation and health status (certified by doctors) of the fetal parents used to establish cell lines, and the three generations of fetal parents should have no obvious history of genetic defects. Before extracting fetal tissue, a detailed investigation should be carried out.
The type of primitive tissue, the number of cells cultured in primitive cells and the growth state.
Cell culture methods, history, growth characteristics and generation life.
Composition of cell growth fluid.