Cell recovery

Cell recovery is to ensure that the extracellular crystals melt in a short time by rapid melting, to avoid the damage caused by the slow melting of water into the cells and recrystallization, and the successful recovery of cells can maintain a high vitality^[1][2][3][4].

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Prepare an ice box before the experiment, quickly remove the frozen cells from the liquid nitrogen, and place them in the ice box.

2. Quickly put the cryopreservation tube into a water bath that has been preheated to 37°C to thaw quickly, and shake it constantly to melt the liquid in the tube quickly. Pay attention that the mouth of the tube is higher than the water surface, so as not to enter and cause pollution.

3. Quickly wipe the outside of the cryovial with an alcohol cotton ball for sterilization and disinfection, and then place it on an ice bath.

4. Take out the cryopreservation tube from the 37°C water bath, open the lid, suck out the cell suspension with a straw, add it to the centrifuge tube and add more than 10 times of fresh culture medium dropwise, mix well; centrifuge at 1000 rpm for 5 min.

5. Discard the supernatant, add more than 10 times fresh culture medium, mix well; centrifuge, 1000 rpm, 5 min.

6. Discard the supernatant, add 10% fetal calf serum culture medium to resuspend the cells, count, adjust the cell density, inoculate the culture dish, and culture in a 37°C incubator.

7. Replace the culture medium the next day and continue the culture.

1. Pay attention to aseptic operation during the whole process of cell recovery.

2. Wear gloves or use tweezers when taking the cells out of the liquid nitrogen tank to prevent frostbite.

3. In the process of cell recovery, the temperature rise rate should be large, and the cells taken out of liquid nitrogen or -70 ℃ refrigerator should be put into the water bath in the shortest time.

4. The thawing process should be within 1 minute to prevent the cells from being damaged by a large number of ice crystals during the thawing process. After about 1 minute, the liquid in the cryopreservation tube is completely dissolved. Extending the water bath time to 37°C will increase the cell death rate, and the general cell death rate is between 20% and 25% during the resuscitation process.

5. When the cells are thawed, it is not advisable to resuscitate too many cells at one time. There are too many cryotubes in the water bath, which will lead to poor heat transfer and prolong the thawing time. Moreover, if there are too many cells recovered at one time, it is easy to forget to replace the tips and pipettes, resulting in cross-contamination of cells.

6. The cryopreservation solution is toxic to the cells. After thawing, it must be washed twice with PBS or culture medium before being transferred to a culture bottle for culture.

7. During the process of taking cells, antifreeze gloves and goggles were not worn. This is particularly important. The cell cryopreservation tube may leak into liquid nitrogen, and the temperature in the cryopreservation tube rises sharply during thawing, which may cause an explosion. Of course, this situation will generally not happen with a good cryopreservation tube.