After a laboratory obtains a new cell line, the cell line must first be cultured and expanded and then frozen. The cryopreservation of cells can firstly be used as a backup when cells are lost due to pollution and other conditions. In addition, almo......
After a laboratory obtains a new cell line, the cell line must first be cultured and expanded and then frozen. The cryopreservation of cells can firstly be used as a backup when cells are lost due to pollution and other conditions. In addition, almost all cells undergo a certain degree of variation during the process of culture and passage. In order to maintain the consistency of experimental results, generally cells cannot be cultured for dozens of generations. If it is used again, it is necessary to thaw and culture the cryopreserved, less passaged cells before use.
There are four main points in the cell freezing process: cell harvesting, use of protective agents, freezing rate and storage environment.
The cells used for cryopreservation are generally selected when the cells are approximately 90% confluent. At this time, the cell growth is good and the number of cells is large, and the culture medium should be changed 24 hours before harvesting the cells. The method of harvesting the cells for cryopreservation is the same as usual. Centrifuge at 100xg for 5 minutes to collect the cells and resuspend them. The live cells are counted. Dilute or concentrate to twice the final cell concentration for cell preservation (usually 4-10 million cells/ml), add an equal volume of the culture medium protective agent mixture solution that has been prepared, and mix gently, and aliquot to cryopreservation Tube, cryopreservation.
Use of protective agents.
In cell cryopreservation, DMSO is generally used as a protective agent. In cell cryopreservation, the final concentration of DMSO used is generally 5-15%. The concentration of DMSO in the cryopreservation solution of a specific cell can be found from ATCC. For particularly sensitive cells, especially hybridoma cells, it may be better to use 95% serum and 5% DMSO when cryopreserving. When using the protective agent, use culture medium or serum to make twice the final concentration, and then mix it with an equal volume of suspension cell culture medium.
Cell freezing rate
The freezing rate of cells is best controlled to allow sufficient time for the cells to be dehydrated without being damaged by excessive dehydration. For most cells, a temperature drop of 1 to 3°C per minute is appropriate. The usual operation is to put the cells at -20°C for 1-2 hours, then put them in the -80°C refrigerator overnight (if there is no -80°C refrigerator, you can use dry ice instead), and then transfer to the liquid nitrogen tank or below -130 Long-term storage in refrigerator at ℃.
The long-term storage temperature of cells is -130 ℃ or lower. The temperature of the gaseous layer in the liquid nitrogen tank is between -140℃ and -180℃. Cells can be stored in the gaseous layer or immersed in liquid nitrogen. If possible, it is best to store in the gaseous layer, because it can avoid liquid nitrogen from entering the freezing storage. The tube causes an explosion when the cells are taken out (but this way, only a small amount of liquid nitrogen can be stored in the liquid nitrogen tank, which needs to be added frequently). The cells can also be stored in the refrigerator at -80℃ for a few months or so.