Cell culture is the basic technology for virus research and vaccine development. Therefore, cell culture technology has been widely used in genetics, immunology, oncology, virology, molecular biology and other fields. Nuclear transplantation, cell hybridization, DNA-mediated gene transfer and the establishment of some physical maps developed in recent years also need to be closely integrated with cell culture.
The general process of cell culture mainly includes the following points:
1. Preparation: The preparation includes the cleaning, drying and disinfection of utensils, the preparation, packaging and sterilization of culture media and other reagents, the cleaning and disinfection of sterile rooms or ultra-clean benches, and the preparation of incubators and other instruments. Check and debug.
2. Extracting material: Take out a certain tissue cell from the body in a sterile environment (depending on the purpose of the experiment), and put it into a culture vessel after a certain treatment (such as digestion and dispersion of cells, separation, etc.). This process is called material extraction. . In the case of cell line expansion, there is no such process as obtaining materials. The first culture of tissue cells taken out of the body is called primary culture.
3. Cultivation: The process of putting the obtained tissue cells into a culture flask or culture plate is called culture. For tissue block culture, connect the tissue block directly to the bottom of the culture vessel. After a few hours, the tissue block can be attached to the bottom, and then add the culture medium.
4. Cryopreservation and resuscitation: In order to preserve cells, especially mutant cells or cell strains that are not easily available, the cells should be frozen. The freezing temperature generally uses the temperature of liquid nitrogen -196℃. Collect the cells into the cryopreservation tube and add a medium containing a protective agent (usually dimethyl sulfoxide or glycerol), and freeze at a certain cooling rate. Store in liquid nitrogen. At J low temperature, the storage time of cells is almost unlimited. The resuscitation generally adopts a quick-thaw method, that is, after removing the cryotube from the liquid nitrogen, immediately put it in 37°C water to make it melt quickly within one minute. Then transfer the cells to a culture vessel for culture.
The basic steps of cell primary culture:
Primary isolation cell culture refers to the separation of tissues from the donor body into single cells or monotype cell groups by mechanical and digestion, so that they can simulate the physiological environment of the human body in vitro, under sterile, appropriate temperature and certain nutritional conditions. Survive, grow and reproduce. Primary cultured cells often have different cell components and grow slowly, but they are more representative of the tissue cell type from which they are derived and the specific characteristics of the expression tissue. The use of primary cell culture for various experiments, such as drug testing, cell differentiation and virology, has a very good effect. The operation steps are as follows:
1. Cut the tissue: First, clean the obtained tissue with D-Hanks or Hanks solution to remove blood stains on the surface, and use surgical forceps to remove the attached connective tissue and other non-cultivating tissues. After cleaning again, use a scalpel to cut the tissue into several small pieces, transfer them into a penicillin vial or a small beaker, add an appropriate amount of buffer, and use elbow ophthalmic scissors to repeatedly cut the tissue until the tissue becomes a paste, about 1mm3 in size. After standing for a while, use a straw to suck up the upper layer of liquid, add appropriate buffer solution and wash again.
2. Digestion and separation: The purpose of digestion and separation is to digest and separate small tissue pieces into cell clusters or scattered individual cells to facilitate further culture. Commonly used digestive enzymes are trypsin and collagenase.
3. Cultivation: Use a counting plate to count the cells of the cell suspension. Adjust the number of cells to (2～5)×105 cells/ml with the culture medium, or the density required for the experiment, and divide them into culture flasks so that the amount of cell suspension is slightly higher than the bottom of the culture flask after covering. Place in a CO2 incubator, 5% CO2, 37°C for static culture. Generally 3~5d, the primary cultured cells can adhere to the bottle wall and stretch to start to grow. You can add 1/2 of the original culture medium to the new culture medium, continue to culture for 2~3d and then change the medium, generally 7~14d can be longer Fill the wall of the bottle and pass it down.
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