Procedures and Precautions for Cryopreservation of Adherent Cells and Suspension Cells

When we need to transport or store cells for a long time, we usually use cryopreservation. If cells are not cryopreserved properly, it will directly affect the state of cell recovery, so cell cryopreservation is very important.

This issue will introduce the cryopreservation procedures and precautions of adherent cells and suspension cells.

Before the cryopreservation operation, the cryopreservation solution can be prepared in advance. If serum-free non-programmed cryopreservations are used, there is no need to prepare the cryopreservation solution and no need for a program cooling box.

Adherent cell cryopreservation

◆ Remove the supernatant from the cells to be operated and rinse with PBS.

◆ Remove the supernatant, add trypsin to dissociate, add complete medium to terminate dissociation after the completed dissociation, and pipette evenly to prepare a cell suspension.

◆ After centrifuging at 1200rpm (250g) for 3min, aspirate the supernatant as clean as possible, and collect the cell precipitate.

◆ Add the prepared cryopreservation solution to resuspend. A T25 culture flask grows to about 80% density of cells can be frozen one cryotube. Or after cell counting, 3~5×10^6 cells/cartridge should be used for cryopreservation, and 0.5~1mL/cartridge of cryopreservation solution is recommended.

◆ After dispensing, tighten the cap of the cryotube and mark it.

◆ Transfer the dispensed cryotubes into the program cooling box and put them into the -80℃ refrigerator overnight.

◆ Finally, transfer the cryotubes to liquid nitrogen for long-term storage.

Suspension cell cryopreservation

◈Centrifuge the cell suspension directly at 1200rpm (about 250g) for 3 minutes, then aspirate the supernatant as clean as possible, and collect the cell precipitate.

◈Remove the supernatant and resuspend the cells with the prepared cryopreservation solution. A T25 culture flask grows to about 80% density of cells can be frozen one cryotube. Or after cell counting, 3~5x10⁶ cells/cartridge should be used for cryopreservation, and 0.5~1mL/cartridge of cryopreservation solution is recommended.

◈After dispensing, tighten the cap of the cryotube and mark it.

◈Transfer the dispensed cryotubes into the program cooling box and put them into the -80℃ refrigerator overnight.

◈Finally, transfer the cryotubes to liquid nitrogen for long-term storage.

Precautions

1. The cell cryopreservation solution should be prepared in advance, and DMSO cannot be directly added to the cell suspension.

2. Cells with a confluence of 80%-90% and in the logarithmic growth phase should be selected for cryopreservation to ensure the best cell state during cryopreservation, and the cryopreservation density can be adjusted according to the characteristics of the cells.

3. Reagents such as program cooling boxes, cell cryopreservation solution, and complete medium must be rewarmed to room temperature for later use.

4. Before cryopreservation, be careful not to dissociate for too long, blow too hard, centrifuge at too high a speed or centrifuge for too long to avoid cell damage.

5. Long-term storage of frozen cells should be placed in liquid nitrogen, and long-term storage at -80°C is not recommended.

6. If there is no programmed cooling box, lower the temperature in the following order: room temperature → 4°C for 20 minutes → -20°C for 30 minutes → -80°C overnight → store in liquid nitrogen. Keep cold during the transfer to avoid temperature difference or melting of the cryotube.