The resuscitation of frozen cells can be used for: (1) In the experimental manipulation of cells, frozen cells are resuscitated and then cultured for passaging. (2) Durable retention of cell lines

Operation method

Resuscitation of frozen cells

Principle

Cryopreserved cells should be thawed quickly under running water and added to the growth medium. Goggles and gloves should be worn by the experimenter because liquid nitrogen may leak from a cryotube that has been submerged in liquid nitrogen, and an increase in the temperature of the gas in the cryotube when thawing the cryopreservative can lead to an explosion.

Materials and Instruments

Frozen Cells
Ethanol Cell Culture Solution
Freezing tubes, water bath

Move

1) Adjust the temperature of the faucet to 40°C. Place a wide-mouth beaker under the faucet.

2) Remove the cryopreservation tube for freezing cells from liquid nitrogen.

3) Place the tube under running water at 40°C until it is completely melted, once the ice is gone remove the tube from the water bath.

The purpose of this step is to thaw the cells as quickly as possible, but not to allow the temperature of the cells to rise above 37°C and once thawed the cells should not be kept in the cryopreservation solution.

4) Scrub the outside of the tube with 70% ethanol to minimize the chance of contamination.

5) Aspirate the contents of the cryotube into a T-25 cell culture flask or dish, slowly add 4 ml of growth medium and mix well.

6) Incubate the cells at 37°C and change the culture medium within 24 hours to nourish the cells.

Caveat

- Some types of cells show better recovery if the culture solution is added slowly to the cryopreservation solution to allow the cryopreservation solution to be removed, and then the cells are centrifuged and resuspended in fresh culture solution.- Freezing Records: It is important to keep accurate records of frozen cells. A history of cell growth needs to be established, name, number of passages, date of freezing, number of resuscitations, growth medium and location in the freezer are all items that should be recorded.- Cryopreservation Solution: Growth medium supplemented with 10%-20% FBS and 10% glycerol or DMSO should be used; DMSO is recommended for most suspension cells. The optimal cryopreservation solution is determined by comparing the concentration of FBS (10%-20%), DMSO (5%-20%), or glycerol (10%-15%). Comparisons of resuscitation efficiency are made after several days of storage at low temperature.Caution: Glycerol; DMSO

Common Problems

Cells are placed at low temperatures to reduce cellular metabolism for long-term storage.

Source "Guide to Cellular Experiments (Previous Book)" by Huang Peitang.

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