Application of dimethyl sulfoxide in cell cryopreservation

Dimethyl sulfoxide is an important osmotic cell protectant. It is necessary to use a cryoprotectant containing DMSO in order to prevent damage caused by the formation of intracellular liquid ice crystals, osmotic pressure changes, and cell structure disorders during the cryopreserved process of cells at a deep temperature (minus 200 degrees Celsius).
DMSO can quickly penetrate the cell membrane into the cell, reduce the freezing point, delay the freezing process, increase the concentration of intracellular ions, reduce the formation of intracellular ice crystals, and thus reduce cell damage.
​
The cytotoxicity of dimethyl sulfoxide is inhibited at deep low temperature, and the action should be fast during resuscitation to wash off dimethyl sulfoxide as soon as possible, otherwise it will cause serious toxicity to cells.
Dimethyl sulfoxide (DMSO) is the best protective agent for cell cryopreserved, but it is also a chemical test agent with high cytotoxicity. The results show that when the concentration of DMSO in culture medium is 10%, the cell growth inhibition rate is nearly 100%. At the concentration of 1‰, the inhibition rate was 35%, and even at the concentration of 0.04‰, DMSO had adverse effects on the growth of cells.

How do you use it in an experiment? I’ll tell you next
01 cell cryopreservation
① Frozen culture solution containing 10%DMSO or glycerin and 10-20% calf serum was prepared.
② Take the cells of logarithmic growth stage, remove the old culture medium, and clean them with PBS.
③ PBS was removed and appropriate amount of trypsin was added (covering the surface of the petri dish) to digest the single layer of growing cells.
④ Centrifuge 1000rpm for 5min.
⑤ Remove trypsin, add appropriate amount of prepared frozen culture solution, gently blow with a straw to make cells uniform, count, and adjust the final density of cells in the frozen solution to 5×106/ml ~ 1×107/ml.
(6) The cells were divided into frozen storage tubes, each tube 1 ~ 1.5ml.
⑦ Indicate the cell name, freezing time and operator on the frozen storage tube.
Freezing: the standard freezing procedure is the cooling rate of -1 ~ -2℃/min; When the temperature reaches below -25℃, it can be increased to -5℃ ~ -10℃/min; At -100 ° C, it can be quickly immersed in liquid nitrogen. Alternatively, the cryopreserved tube containing cells can be placed in the -20℃ refrigerator for 2h, and then placed in the -70℃ refrigerator overnight, and the cryopreserved tube can be removed and transferred into the liquid nitrogen container.
02 Cell recovery
① Remove the frozen storage tube from the liquid nitrogen container, directly immerse in 37℃ warm water, and shake it from time to time to melt as soon as possible.
② Remove the frozen storage tube from the 37℃ water bath, open the lid, suck out the cell suspension with a straw, add it to the centrifuge tube, add more than 10 times the culture solution, and mix well.
③ Centrifuge, 1000rpm, 5min.
④ Discard the supernatant, add the culture medium containing 10% calf serum to resuspension cells, count, adjust cell density, inoculate the culture bottle, and incubate at 37℃.
⑤ The next day, replace the culture medium and continue to culture.
So that’s DMSO
Methods of use in cell cryopreservation
DMSO and other kinds of chemical raw materials, reagents are available for sale