Eight things to pay attention to in cell cryopreservation

In the process of passage and daily maintenance of cell culture, a lot of consumption is required in terms of culture utensils, culture medium and various preparations, and once the cells leave the living body to start primary culture, its various biological properties will gradually change and With the increase of the number of passages and changes in the in vitro environmental conditions, there are new changes
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Therefore, timely cryopreservation of cells is necessary
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Cells can be stored frozen at -70°C for up to one year; cells are stored in liquid nitrogen at a temperature of -196°C, and the storage time is theoretically unlimited
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The following are the precautions for cell cryopreservation: 1.
In order to maintain the maximum cell viability, the method of slow freezing and thawing is generally adopted
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2.
The closer the frozen material is to the liquid nitrogen surface, the lower the temperature
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The standard low freezing speed is -1℃~12℃/min.
When the temperature drops to -25℃, the descending speed can be increased to -5~-10℃/min.
When it reaches -100℃, it can be quickly frozen into liquid nitrogen.
in
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Therefore, it is necessary to properly grasp the speed at which the cryopreserved material falls into the liquid nitrogen.
Too fast will affect the permeation of water in the cells, and too slow will promote the formation of ice crystals
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3.
For the first-time cryopreserved, when descending cryopreservation, it is advisable to use a thin wooden stick to extend into the tank to detect the position of the liquid level, and then use a thermometer in parallel with the cryopreservation to observe the speed of descending cryopreservation and the cryopreservation.
The relationship between the distance and the temperature of the liquid nitrogen and the liquid nitrogen surface, when the above parameters are mastered and the cryopreservation technique is proficient, the ideal cryopreservation effect can be obtained only by the descending time and descending distance
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4.
Different cells have different requirements for cryopreservation speed; epithelial cells and fibroblasts may be more tolerant, and bone marrow stem cells -2~-3℃/min are suitable; embryonic cells are less tolerant and should not be too tolerant.
fast
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In short, at the beginning, the descending speed cannot exceed -10°C/min
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5.
The appropriate amount of protective agent to use depends on the cells.
It is better to use DMSO for primary cultured cells and glycerol for general cells; the smaller amount is better
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Some people think that human skin epithelial cells are well stored in 20%-30% glycerol
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6.
In principle, cells can be stored in liquid nitrogen for many years, but for the sake of safety, after one year of cryopreservation, the cells should be revived and cultured again, and then continue to be cryopreserved
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7.
When putting the cryovial into or out of the liquid nitrogen container, take precautions to avoid frostbite.
8.
Pay attention to your own safety, and be especially careful with cell lines derived from human or virus infection
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During the operation, avoid the generation of aerosols, be careful with toxic reagents such as DMSO, and avoid hurting people with sharp objects
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