Method for quickly cryopreserving and thawing cells

Abstract

The invention provides a method for quickly cryopreserving and thawing cells, which comprises a cryopreservation step and a thawing step. The cryopreservation step comprises: (1) cells are dissociated, and cell suspension is prepared; (2) the cells are diluted and separately packaged; (3) temperature is gradiently decreased, and is increased, temperature is then gradiently decreased to negative 120 DEG C to negative 180 DEG C, and the cells are stored in liquid nitrogen. The thawing step comprises: the cryopreserved cells are taken out of the liquid nitrogen, quickly immersed in thawing waterwhich is 38 DEG C to 40 DEG C and rapidly shaken until the cells are completely thawed, and the cells can be directly used without removing the cryopreserving liquid. The method disclosed by the invention can guarantee the quality consistency between batches of cryopreserved cells and the stability of cell genetics, ensure that the survival rate of thawed cells can be stably increased to 98 percent or more, and shorten the cell thawing and passage period by 50 to 60 percent.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for rapidly freezing and resuscitating cells. Background technique [0002] With the development of cell biology application technology, in vitro cell culture technology has been widely used in various biological research and industrial production fields. Therefore, cell cryopreservation and recovery technology is one of the key areas of cell culture technology. How to establish an efficient cell cryopreservation method to ensure long-term biological activity of cryopreserved cells and improve cell survival rate during cell recovery is a technical difficulty often encountered in current cell biology experiments and some industrial production. [0003] A large number of studies have shown that storing cells in liquid nitrogen at -196°C can preserve their characteristics for a long time and make the cells temporarily out of the growth state. Afterwards, the cells can be revived...

AI Technical Summary

Problems solved by technology

[0007] The characteristics of the cell resuscitation technology in the prior art are (1) resuscitating cells at low temperature (37°C water bath), the recovery time is too long, and the cells are easily damaged during the rewarming process

(2) Resuscitate cells at high temperature (46-67°C water bath). Although the recovery time is shortened, the cells are easily damaged during the rewarming process.

(3) The influence of other solution temperatures on the recovery survival rate was not considered during the cell recovery process

(4) After the cells are resuscitated, they need to be centrifuged at a low speed in a centrifuge (800 rpm, centrifuged for 5 minutes) to remove the cryopreservation solution, and then dilute the cells collected by centrifugation and put them into use, which is easy to cause secondary damage to the cells after resuscitation

[0008] The above methods are all in line with the principle of rapid recovery of cells to reduce the formation of intracellular crystals, and the general requirements of cell recovery have been achieved, but the impact of freezing rate and recovery rate on cells has not been fully considered.

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