When the temperature is below -70 ℃, the enzyme activity in the cells has stopped, that is to say, the metabolism is completely stopped, so it can be preserved for a long time. The key of cell cryopreservation is through the process of 0~20 ℃.In this temperature range, the crystal is needle like, which is easy to cause serious cell damage. The general principle is slow freezing.
Cultured cells with good growth,fresh medium,DMSO,sterile plastic cryopreservation tube,blood cell counting disk and cover slide.
1. One day before freezing, change half or full amount of medium to observe cell growth. The best cell should be in logarithmic growth period.
2. Preparation of cryopreservation solution (preparation before use):DMSO was added to the fresh medium, the final concentration was 5-10%, mixed evenly, and placed at room temperature for use.
3. According to the cell subculture operation, collect the cultured cells and cryopreservation tube, take a small amount of cell suspension (about 0.1 ml) to count the cell concentration.
4. Put the cryopreservation tube into a 4 ℃ refrigerator for about 40 minutes.
5. Then put it in the refrigerator of - 20 ℃ for about 30-60min.
6. Overnight in -80 ultra low temperature refrigerator.
7. Stored in liquid nitrogen tank for a long time.
8. At the same time, make a record of frozen storage, which should be recorded in your own notebook and frozen storage record book.
1. Before using DMSO, it does not need to be autoclaved, it has the function of sterilization itself.On the contrary, autoclave will destroy its molecular structure and reduce its cryoprotective effect. At room temperature, DMSO is harmful to human body, so it is better to wear gloves when preparing.DMSO should be mixed quickly, because DMSO is toxic to cells. After mixing, it should be frozen as soon as possible. Please note that it must be mixed well after adding the frozen solution to prevent DMSO precipitation.
2. The cryopreserved cells should not be placed in the temperature range of 0 ℃ ~ -60 ℃ for too long. The low temperature damage mainly occurs in this temperature range, which is a "dangerous temperature zone". You can first punch holes in the foam and plug in the freezing pipes.It can slow down the temperature. Of course, it's a good idea to pack cotton.
3. Pay attention to regularly check the liquid nitrogen quantity in the liquid nitrogen tank and add it in time.
4. -20 ° C can not exceed 1 hour to prevent cell death due to excessive ice crystals. This step can also be skipped. The cryotube is placed in the styrofoam and directly put into the refrigerator at -80°C, but the survival rate will be reduced.
5. The number of cells in the freezing tube is generally more than 10 6-7 / ml,the healthy degree of cryopreserved cells generally requires that the living cells are more than 95%. The survival rate of cells with poor viability is very small after cryopreservation. Therefore, cryopreservation must be carried out in the period of vigorous cell division
6. Generally, the cell cryopreservation tube has a volume of about 1.5-2ml. In principle, it can store 1.5ml of cryopreservation solution filled with evenly suspended cells. Some people like to pour 1-1.5ml of cryopreservation solution for cryopreservation, which is not good in fact.The reasons are as follows: It takes a period of time for the frozen storage liquid to be frozen. If the frozen storage tube is inclined during this period of time, the liquid is easy to flow to the nozzle, and it is easy to cause pollution in the subsequent operation. It is suggested that 0.5-1ml.However, if 1.5ml is still used, during the specific operation, the frozen storage tube shall be upright and placed in the refrigerator of -20, which is not easy to cause pollution. At the same time, when freezing and storing cells, the sealing film must be added, so as to avoid the water in the water bath box entering into the gap of the lid during the recovery. After the freezing storage tube is sealed with sealing film, it is better to seal it with white tape. In this way, when the cells are revived, the sealing film will crack and the water in the water bath box will enter the cryopreservation tube.
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