1. Cell cryopreservation is a technology that stores cells in a low-temperature environment to reduce cell metabolism and achieve long-term storage.
2. In most cell lines, cells will continue to accumulate changes with aging and evolution, resulting in "culture drift" of phenotypes and genotypes. During the cryopreservation process, appropriate operations and Appropriate cryopreservation conditions can reduce the change or loss of cell characteristics and play a role in cell species preservation. Therefore, correct and successful cryopreservation plays a very important role in the long-term use of cells.
1. When the temperature is as low as -70°C, all enzyme activities in the cells stop and the metabolic activity stops, so the purpose of long-term preservation is achieved.
2. As the temperature decreases, the water inside and outside the cells will crystallize. The ice crystals formed will cause damage to the cell membrane and organelles, thus causing cell death. Especially at the 0~-20℃ stage, the ice crystals appear needle-like, and causes cell damage. When cells are directly frozen without any protective agent, water in the cells and in the external environment will form ice crystals, which can cause mechanical damage, increased electrolytes, changes in osmotic pressure, dehydration, changes in pH, and protein denaturation in the cells. , can cause cell death.
3. Common cryoprotectants are glycerol or dimethyl sulfoxide (DMSO). DMSO can quickly penetrate the cell membrane and enter the cells, lower the freezing point, delay the cryopreservation process, increase the permeability of the cell membrane, increase the intracellular ion concentration, and reduce the formation of intracellular ice crystals, thereby achieving the purpose of protecting cells.
1. In cell cryopreservation, DMSO is generally used as a protective agent. In cell cryopreservation, the final concentration of DMSO used is generally 5-15. The DMSO concentration in the cryopreservation solution for a specific cell can be found from the ATCC.
2. For particularly sensitive cells, it is necessary to test the appropriate freezing solution when freezing them. For example, when freezing neuronal cells, it is necessary to choose a serum-free cryopreservation solution to avoid differentiation after recovery.
·?Maintenance of limited cell line morphology and metabolic activity
·?Preservation of precious cell species
·?Preparation of cell line standards
·? Umbilical cord blood stem cells and NK cells cryopreservation
1. The cells used for cryopreservation are generally selected in the logarithmic growth phase of the cells, when the confluency is about 90, when the cells are in good growth status and the cells The quantity is also large, and the culture medium is changed 24 hours before harvesting cells.
2. Test the culture for microbial contaminants, especially mycoplasma, to ensure that the cells are free of any contamination. During the cell collection process, experimental operations should be as gentle as possible to avoid cell damage.
1. The freezing rate of cells is optimally controlled so that the cells have enough time to dehydrate without being overly dehydrated and causing cell damage. For most cells, a decrease of 1 to 3°C per minute is appropriate.
2. Gradient cooling method: 4℃-30 min, 20℃-2h, freeze overnight in -80℃ refrigerator, and transfer the cryopreservation tube to a liquid nitrogen tank for long-term storage the next day.
3. Cell programmed cooling box, put the prepared cell cryopreservation tube into the cryopreservation box, and then transfer it to the liquid nitrogen tank directly after leaving it at -80°C overnight.
4. The transfer to the storage location must be done quickly. It is recommended to place the cryovials in dry ice at a constant temperature during the transfer to avoid the impact of temperature changes on cell viability during the transfer process.
1. Cells can also be stored in a -80°C refrigerator for several months.
2. The long-term storage temperature of cells is -130 ℃ or lower.
3. The temperature of the gaseous layer in the liquid nitrogen tank is between -140℃ and -180℃. Cells can be stored in the gaseous layer or immersed in liquid nitrogen. If possible, it is best to store them in the gaseous layer because this It can avoid the explosion caused by liquid nitrogen entering the cryovial when taking out the cells (but only a small amount of liquid nitrogen can be stored in the liquid nitrogen tank, and it needs to be added frequently).
HyCyte? cryopreservation solution is a ready-to-use, serum-free, "long-term" cell cryopreservation solution that does not require programmed cooling. The product formula has been used for more than 15 years. The raw materials are imported from Japan. The stock quality is consistent with Japanese products.
·? Safe: serum-free, no xenogeneic animal ingredients
·? Broad spectrum: suitable for a variety of cell types
·? Efficient: cells after recovery Maintain high activity and rapid proliferation ability without affecting cell function
· Convenient: Ready-to-use, no additional preparation, no need for programmed cooling box and dilution
Cell membrane protectant - can A protective film is instantly formed outside the cell membrane to protect cells from damage by extracellular ice crystals.
Osmotic protective agent—can quickly penetrate into cells to prevent the production of large amounts of ice crystals inside cells. It can also protect organelles and reduce damage at low temperatures.
Cell sedimentation stabilizer—can balance the cell sedimentation speed and prevent cells from aggregating into clumps during cryopreservation.