Cell cryopreservation:

1. When preparing DMSO, it will release heat and must be used after the cryopreservation solution cools down to avoid burning cells; 

2. After cell centrifugation, try to aspirate the supernatant as clean as possible to reduce residual culture medium and avoid diluting the cryopreservation solution; 

3. It is not recommended to manually gradient cool down because the temperature is unstable and can easily reduce the survival rate; 

4. The amount of isopropanol in the program cooling box must be higher than the minimum mark; After 5 freezes, isopropanol needs to be replaced once to avoid affecting the freezing effect; The program cooling box needs to be restored to room temperature before it can continue to be used, and cannot be taken out of the refrigerator for direct use; 

5. After adding the cell suspension to the cryopreservation solution, avoid prolonged storage at room temperature, as DMSO can cause significant damage to cells at room temperature; After the packaging is completed, immediately transfer to the program cooling box and place it in the -80 ℃ refrigerator, without the need for a 4 ℃ refrigerator; 

After being frozen at -80 ℃ overnight, it can be transferred to liquid nitrogen for long-term storage; The process of transferring to liquid nitrogen requires keeping the freezer box cold and not placing it at room temperature for a long time to prevent the freezer tube from melting; 

7. If there is no liquid nitrogen tank, when stored at -80 ℃, it must be placed inside to avoid temperature instability caused by turning on or off the refrigerator; 

8. After cell cryopreservation, one tube should be taken out for recovery and the cell survival rate should be tested. In theory, cells can be stored in liquid nitrogen for a long time. For safety reasons, cells can be revived and cultured after being frozen for six months to observe their growth, and then continue to be frozen; 

9. If a cryopreservation box is not used, it is necessary to take cold insulation measures for the cryopreservation tube during the transfer of liquid nitrogen. It cannot be directly held by hand, but can be protected with dry ice or a small amount of liquid nitrogen before transfer. Pay attention to safety during the operation; 

10. The transfer process should be fast to prevent the frozen storage tube from melting after being exposed to room temperature.

Cell Resuscitation:

If the water bath and liquid nitrogen tank are not placed in the same room, the cryopreservation tube needs to be kept cold before being transferred to the water bath to avoid melting of the cell surface along the way; 

2. The process of cell dissolution should be rapidly shaken to accelerate dissolution; 

3. Dissolved frozen cells should be avoided from being stored at room temperature for a long time, and DMSO should be removed by centrifugation as soon as possible; 

4. After 24 hours of cell recovery, observe the adherent cells. If the density reaches 80%, the cells are passaged normally; If the density is less than 80%, continue to cultivate for 48 hours before changing the solution; 

5. It is not recommended to change the centrifugation solution within 3 days of suspension cell recovery; 

6. Cells that are not sensitive to DMSO may not be centrifuged during recovery; Reducing operational steps can also reduce the likelihood of contamination. Transfer the thawed cell suspension directly to a T25 cell bottle, add fresh culture medium, and place it in a incubator for cultivation. But after 12-24 hours of cultivation, fresh culture medium must be replaced and dead cells removed.