Cryopreservation of cells is one of the main methods of cell preservation. Using cryopreservation techniques to store cells in cryopreservation at -196 °C in liquid nitrogen allows the cells to be temporarily detached from the growth state and preserve their cellular characteristics, so that the cells are resuscitated for experimentation when needed. Moreover, by properly storing a certain amount of cells, it is possible to prevent the cells from being contaminated due to contamination or other unexpected events of the cells being cultured, thereby playing a role in cell conservation. In addition, certain cells can be purchased, sent, exchanged, and shipped in the form of cell cryopreservation. In the process of passage and daily maintenance of cell culture, a large amount of labor is required in the culture apparatus, the culture solution, and various preparation work, and once the cells are left from the living body and the primary culture is started, its various biological characteristics will gradually change and There are new changes as the number of passages increases and the environmental conditions in vitro change. Therefore, timely cryopreservation of cells is necessary. The cells are stored frozen in a -70 ° C refrigerator for one year; the cells are stored in liquid nitrogen at a temperature of -196 ° C, and the theoretical storage time is unlimited. Cryopreservation of cells 1. In order to ensure the good state of the cells, the cells are in a logarithmic growth period on the day before the cells are frozen, and the cells are changed. The next day, according to the cell subculture method, the adherent cells are digested with trypsin, and the cells are used. The medium was washed off, and then centrifuged with a 50 mL apex centrifuge tube at 1000 rpm for 4 min, and the supernatant was discarded to collect the cells. 2. Add appropriate amount of cryopreservation solution (10% DMSO and 90% IMDM medium containing 20% ​​serum). Shanghai Chuangsai Technology provides MRS broth liquid medium, product number: C010384-1-250g, price 280 yuan. 3. Dispense into the cryotube, mark it, indicate the cell name, storage time, medium used, etc. Shanghai Chuangsai Technology provides cell cryotubes, Freezing Tubes, product number: C81-FCT002005-0.5ml, 5000/box, price 3272 yuan. 4. Place at 4 ° C for 30 min; -20 ° C for 1.5 h; -70 ° C for 12 h; finally move to a liquid nitrogen tank. Cell recovery The principle of cell resuscitation is rapid melting, because if it melts slowly, the water entering the cell will form ice crystals and cause damage to the cells. Therefore, when resuscitating the cells, the frozen cells are directly placed in a 37 ° C water bath to make them Dissolve quickly. Pour the cell suspension in the cryotube directly into a small flask or petri dish in a clean bench, then add 3 mL of the culture solution. The next day, observe cell growth and replace the cell culture medium. The basic principle of cell cryopreservation and resuscitation is slow freezing and rapid melting, which has been shown to maximize cell viability. At present, cells are cryopreserved by using glycerol or dimethyl sulfoxide as a protective agent. These two substances can improve the permeability of the cell membrane to water, and the slow freezing can cause the water inside the cells to ooze out of the cells and reduce the intracellular ice crystals. Formation, thereby reducing cellular damage due to ice crystal formation. Resuscitation cells should be rapidly melted to ensure that extracellular crystallization is melted in a short period of time, avoiding the infiltration of water into cells to form intracellular recrystallization due to slow melting. CANSPEC quality provides excellent performance, its excellent quality to meet the various needs of your different experiments, spot, product details, scientific instruments, laboratory supplies, Shanghai Chuangsai, mass products special promotions, welcome to inquire!
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Cell cryopreservation and resuscitation methods