Cell cryopreservation is one of the most important methods for cell survival. Using cryopreservation skills to place cells in -196~C liquid nitrogen for low temperature survival, cells can temporarily leave the growth state and survive their cellular characteristics, so that the necessary time to wake up the cells for implementation. And the moderate survival of a certain amount of cells can prevent the cells from being polluted or other unexpected changes and make the cells lose seed, which plays the role of cell preservation. In addition, cell cryopreservation can be used to purchase, send, exchange and transport certain cells. When the cell is frozen, the shielding agent - glycerol or dimethyl sulfoxide (DMSO) with a final concentration of 5%-15% can make the freezing point of the solution low, and in the slow freezing condition, the water in the cell is exposed, eliminating the formation of ice crystals, so as to prevent cell damage. The scale freezing rate starts at -1 to -2℃/min, can be accelerated when the temperature is below -25℃, and can be directly put into liquid nitrogen (-196℃) after -80℃.

The commonly used shielding agent is dimethyl sulfoxide (DMSO) and glycerol, which has small molecular weight, large solubility and easy penetration of cells.

Execution material

Instruments: liquid nitrogen tank, frozen storage tube (plastic screw special frozen storage tube or ampoule bottle), centrifugal tube, straw, centrifuge, etc

Reagents: 0.25% pancreatic enzyme, formation base, formation base containing shielding agent (i.e., frozen liquid)

P.S. Preparation of frozen liquid: The base of glycerol or DSMO, so that the final concentration of 5-20%. The type and dosage of shielding agent vary according to different cells. Survive at 4℃ after preparation.

Pace of implementation

1. Digest the cells and transfer the cell suspension network to the centrifuge tube.

Centrifuge at 2.1000rpm for 10 minutes and discard the supernatant.

3. The formation, counting, and mediation of precipitation and covering liquid to about 5106/ml.

4. Divide the suspension into the frozen storage tube, 1ml per tube.

5. Seal the frozen tube mouth tightly. If the ampoule bottle is used, the flame is sealed, the sealing must be strict, otherwise it is easy to burst when waking up.

6. Label with cell type and frozen date. A metal weight and a string are tied to the outside of the frozen storage tube.

7. Cooling in the following order: room temperature →4 ° C (20 minutes)→ refrigerator freezer (30 minutes)→ low temperature refrigerator (-30 ° C 1 hour)→ gaseous nitrogen (30 minutes)→ liquid nitrogen.

Careful: When operating should be vigilant to avoid liquid nitrogen frostbite. Liquid nitrogen is inspected regularly, supplemented at any time, absolutely not volatile clean, as usually 30 liters of liquid nitrogen can be used for 1 to 1.5 months.