Primary and subculture of cells in vitro, as well as cryopreservation and recovery 1. Experimental principle Cell culture can be divided into primary culture and subculture. Tissue cells obtained directly from the body are cultured for the first time as primary culture; when the primary cultured cells proliferate to a certain density, they need to be re-cultured. After the cultured cells are dispersed, the ratio is 1: 2 or other ratio from a container Transfer to another or several containers to expand the culture. For subculture, the cumulative number of subcultures is the number of cells. The basic principle of cell cryopreservation and recovery is slow freezing and quick thawing, and experiments have shown that this can maximize the preservation of cell viability. At present, glycerin or DMSO is used as a protective agent for cell cryopreservation. These two substances can improve the permeability of the cell membrane to water, and the slow freezing can make the water inside the cell seep out of the cell and reduce the formation of ice crystals in the cell. Reduce cell damage due to ice crystal formation. Resuscitated cells should be rapidly thawed to ensure that the extracellular crystals melt within a short period of time, avoiding the infiltration of water into the cells due to the slow melting and the formation of intracellular recrystallization to damage the cells. 2. Experimental methods material: Mice, physiological saline, 100ml sterilized beaker, 15ml centrifuge tube, petri dish, dropper, sterile forceps, scissors, screen, foam plate, pin, alcohol lamp, culture bottle, culture medium, PBS, 0.25% pancreatin , Ultra-clean workbench, carbon dioxide incubator, inverted microscope, microscope, counting plate, centrifuge, constant temperature water bath, refrigerator (4 ℃, -20 ℃, -70 ℃), liquid nitrogen tank, cryopreservation tube, cryopreservation solution , Waste tank, etc. method: (1) Primary culture: 1. After removing the mouse, drain the alcohol, place it in the ultra-clean table, and fix it on the foam board. 2. Lift the skin with tweezers, cut a transverse incision of the skin with anatomical scissors, tear the skin upward, and then cut the muscles, etc., expose the abdominal cavity, find the spleen on the left side, remove the spleen with elbow ophthalmic forceps, and place it in the In a petri dish. 3. Wash the removed spleen multiple times with normal saline, and remove excess and useless tissue. 4. Place the sieve in a flat dish, place the spleen on the sieve, use tweezers with elbow tweezers, and gently grind on the sieve. At the same time, the culture medium without serum is added dropwise to rinse. 5. Aspirate the milled cell suspension into a centrifuge tube, centrifuge (1000rpm, 5min), and aspirate the supernatant (remove blood, etc.). 6. Add 10ml of culture solution, mix by pipetting, sample and count. 7. Dispense the diluted cell suspension into culture flasks, gently shake to mix, and place on the culture flasks. Make a mark on the face, indicating the cell, group and date. Then put the culture bottle in a carbon dioxide incubator for cultivation. (2) Subculture: 1. Observe the cell morphology under an inverted microscope to determine whether the cells need to be passaged. 2. After opening the culture fluid bottle, pass the alcohol lamp flame and place it around the alcohol lamp flame. 3. Take out the straight dropper, put on a rubber tip, overheat, and put it into the culture bottle. 4. Open the culture bottle, the bottle mouth is overheated, pour the culture liquid in the culture bottle into the waste liquid tank, and wash away the remaining old medium with 2-3mL PBS. 5. Add 0.25% trypsin to the culture flask. The dosage is preferably covered with a thin layer. Digest at 37 ° C. When the inverted microscope is observed and the cells are retracted, the culture flask is immediately turned over to remove the cells from the pancreatin The enzyme is discarded and a small amount of fresh medium containing serum is added. 6. Take the elbow dropper to repeatedly blow the cells off the wall and disperse them. Then add a certain amount of fresh medium containing serum according to the number of sub-passage bottles to make a cell suspension, and then distribute into new culture bottles. Put the bottle cap on, tighten it slightly, and then turn it slightly to facilitate the entry of CO2 gas. Put the culture bottle back into the CO2 incubator. 7. For semi-adherent cultured cells, without pancreatin, pipette directly, add fresh medium, and then aliquot into each bottle. (3) Frozen storage: 1. Aspirate the cell suspension after passage, centrifuge, remove the culture solution, add the cryopreservation solution, and aliquot the cryopreservation tube (the number of cells in the cryopreservation tube is generally (5 ～ 10) × 106 cells / ml, in the 2ml cryopreservation tube) Generally put 1 ~ 1.5ml cells). 2. Freeze in steps o Cryopreservation method 1: The standard freezing procedure is the cooling rate -1 ～ -2 ℃ / min; when the temperature is below -25 ℃, it can be increased to -5 ～ -10 ℃ / min; when it reaches -100 ℃, It can be quickly immersed in liquid nitrogen. o Cryopreservation method 2: Place the cryotube in the program cooling machine with the set program and lower it by 1 ～ 3 ℃ to -80 ℃ every minute, then put it in liquid nitrogen for long-term storage. (4) Recovery: 1. Take out the freezing tube, immediately put it in a 37 ° C water bath and quickly thaw it. Gently shake the freezing tube to make it melt within 1 minute, and move it into the sterile operating table. 2. Open the cryotube and suck the cell suspension into the centrifuge tube. 3. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. 4. After adding the appropriate culture medium, transfer the cells to a culture flask and culture at 37 ° C. 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