1. Preheat the water bath to 37℃ and get a clean disposable PE glove ready for use. In the biosafety cabinet, get a 15mL sterile centrifuge tube, and add 9mL of sterile medium to it.
2. Take out the frozen cells from the liquid nitrogen tank. Put the cryogenic vial into the PE glove, submerge the glove-protected vial quickly in the water bath, and shake it constantly to speed up the melting. Thaw the cells thoroughly in 1 minute.
3. Add the cell suspension to the former prepared centrifuge tube with medium and mix gently.
4. Centrifuge the cells at 1200rpm/min (approximately 250g) for 3 minutes. After centrifugation, we will see cell pellets at the bottom of the tube.
5. Pipette the supernatant and discard it. Add proper medium to resuspend the cells, and gently pipette to mix.
6. After fully mixing, transfer the cell suspension to a T25 cell culture flask. Remember to replenish medium if necessary.
7. Observe the cell status and seeding density under a microscope.
8. Finally, put the flask into the incubator for culture, and make sure the water tray, CO2 concentration, and temperature are in the right condition.
1. If the water bath and liquid nitrogen tank are not in the same room, it is necessary to put the cells in freezing condition when transferring them to the water bath, to avoid melting on the vial surface.
2. During the thawing process, shake quickly to accelerate the melting. So that the cells can pass through -5～0℃ as soon as possible, reducing the probability of cell damage. Thaw the cells thoroughly in 1 minute.
3. For melted cells, minimize the time at room temperature and centrifuge as soon as possible to remove DMSO.
4. For adherent cells, do not observe frequently after resuscitation. The frequent movement will affect the adhesion. It is recommended to observe the resuscitated cells under a microscope after 24 hours. If the density reaches 80%, we can conduct the passage operation. If the density is less than 80%, continue to culture until 48 hours before changing the medium.
5. For suspension cells, it is not recommended to change the medium by centrifugation within 3 days. We can replenish the medium or half change the medium to supplement the nutrition.
6. Cells that are not sensitive to DMSO do not need to be centrifuged during resuscitation. Reducing the operation steps can lower the chance of contamination.
The operation is as follows:
·Transfer the thawed cells to the T25 flask.
·Supplement the fresh medium and then culture the cells in the incubator.
·Refresh medium after 12~24 hours to remove dead cells.