Regarding the steps of cell cryopreservation and recovery, as well as the use of cell cryopreservation solution
Cell cryopreservation is a technique that places cells in a low-temperature environment to reduce cellular metabolism and facilitate long-term storage. This allows cells to temporarily detach from their growth state and preserve their cellular characteristics for use in experiments when needed. Moreover, moderately storing a certain amount of cells can prevent the loss of cells due to contamination or other unexpected events during cultivation, playing a role in cell conservation.
Cell cryopreservation steps
1. Prepare a frozen culture medium containing 10% DMSO or glycerol and 10-20% calf serum;
2. Take cells in logarithmic growth phase, remove old culture medium, and wash with PBS.
3. Remove PBS and add an appropriate amount of trypsin (covering the surface of the culture dish) to digest the monolayer grown cells;
4. Centrifuge at 1000rpm for 5 minutes;
5. Remove trypsin, add an appropriate amount of prepared frozen culture medium, gently blow with a pipette to make the cells uniform, count, and adjust the final density of cells in the frozen culture medium to 5 × 106/ml to 1 × 107/ml;
6. Divide the cells into cryovials, with 1-1.5 ml per vial;
7. Mark the cell name, freezing time, and operator on the cryovial;
8. Freezing: The standard freezing procedure is a cooling rate of -1 to -2 ℃/min; When the temperature reaches below -25 ℃, it can be increased to -5 ℃ to -10 ℃/min; At -100 ℃, it can be quickly immersed in liquid nitrogen. Alternatively, the cryovial containing cells can be placed in a -20 ℃ freezer for 2 hours, then placed overnight in a -70 ℃ freezer, removed from the cryovial, and transferred into a liquid nitrogen container.
Cell recovery steps
1. Remove the cryovial from the liquid nitrogen container and immerse it directly in 37 ℃ warm water, shaking it from time to time to melt it as soon as possible.
2. Remove the cryovial from the 37 ℃ water bath, open the lid, use a pipette to aspirate the cell suspension, add it to the centrifuge tube and drip more than 10 times the culture medium, mix well;
3. Centrifuge at 1000rpm for 5 minutes;
4. Discard the supernatant and resuspend the cells in a culture medium containing 10% calf serum. Count and adjust the cell density. Inoculate the cells into a culture bottle and incubate in a 37 ℃ incubator;
5. Change the culture medium the next day and continue cultivation.
Application of cell cryopreservation solution
1. Serum free cell cryopreservation solution is used for the storage of hematopoietic stem cells, mesenchymal stem cells, and other stem cells.
2. Serum free cell cryopreservation solution is used for the storage of immune cells such as T lymphocytes and NK cells.
3. Serum free cell cryopreservation solution is used for the storage of other types of mammalian cells.
Precautions for cell cryopreservation
1．The cultured cells from the proliferation phase to the formation of dense monolayer cells can be used for cryopreservation, but it is best to use logarithmic growth phase cells. It is best to change the culture medium once a day before freezing;
2．When placing or removing cryovials into liquid nitrogen containers, protective measures should be taken to prevent frostbite;
3. It is best to use newly prepared culture medium for cryopreservation and recovery.
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