What is the formula of cell freezing medium?

With the continuous development of science and technology, medical technology is also constantly improving. The incident of freezing humans a few years ago opened the world's eyes. It turns out that not only food can be frozen and preserved, but even the human body can be frozen and preserved. If you want to achieve this goal, you need cell cooling fluid. How to configure this thing? Let's take a closer look at what is the formula of cell freezing fluid?

(I) Cell cryopreservation

1. Prepare freezing culture medium containing 10% DMSO or glycerol and 10-20% calf serum;

2. Take cells in the logarithmic growth phase, remove the old culture medium, and wash with PBS.

3. Remove PBS and add appropriate amount of trypsin (covering the surface of the culture dish) to digest the monolayer of cells;

4. Centrifuge at 1000 rpm for 5 min;

5. Remove trypsin, add an appropriate amount of prepared freezing culture medium, gently blow with a pipette to make the cells uniform, count, and adjust the final density of cells in the freezing medium to 5×106/ml～1×107/ml;

6. Dispense the cells into cryopreservation tubes, 1-1.5 ml per tube;

7. Label the cell name, freezing time and operator on the cryopreservation tube;

8. Cryopreservation: The standard cryopreservation procedure is a cooling rate of -1 to -2°C/min. When the temperature reaches below -25°C, it can be increased to -5°C to -10°C/min. When it reaches -100°C, it can be quickly immersed in liquid nitrogen. Alternatively, the cryotubes containing cells can be placed in a -20°C refrigerator for 2 hours, then placed in a -70°C refrigerator overnight, the cryotubes can be taken out, and placed in a liquid nitrogen container.

(II) Cell recovery

1. Take out the cryovial from the liquid nitrogen container and immerse it directly in 37℃ warm water, shaking it from time to time to melt it as quickly as possible.

2. Take out the cryotube from the 37℃ water bath, open the lid, suck out the cell suspension with a pipette, add it to the centrifuge tube and add 10 times more culture medium, mix well;

3. Centrifuge, 1000 rpm, 5 min;

4. Discard the supernatant, add culture medium containing 10% calf serum to resuspend the cells, count, adjust the cell density, inoculate the culture flask, and culture in a 37°C incubator;

5. Replace the culture medium the next day and continue culturing.

Precautions

1. Cultured cells from the proliferation stage to the formation of a dense monolayer can be used for cryopreservation, but cells in the logarithmic growth phase are preferred. It is best to change the culture medium once a day before freezing;

2. When placing the cryotube into or taking it out of the liquid nitrogen container, take protective measures to avoid frostbite;

3. It is best to use freshly prepared culture medium for freezing and thawing.