Programmed cooling method for human neural stem cell working cell bank

A technology of programmed cooling and stem cells, which is applied in the preservation, application, and animal husbandry of human or animal bodies. It can solve the problems of cell damage, low cell survival rate, and poor synergy, and achieve high activity effects.

Pending Publication Date: 2021-04-20
华夏源细胞工程集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] When neural stem cells are cryopreserved, the selection of cryopreservation medium and the step of programmed cooling are very critical steps. However, the synergy between the selection of cryopreservation medium and the setting of programmed cooling in the prior art is not very good. Can cause cells to be damaged during cryopreservation, resulting in lower cell viability after thawing

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  • Programmed cooling method for human neural stem cell working cell bank

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Embodiment 1

[0082] Embodiment 1 of the present invention specifically provides a method for programmed cooling of a working cell bank of human neural stem cells. The steps include: (1) preparation of cryopreservation solution: adding protective agent to the centrifuge tube, shaking and mixing the culture medium, adding nutrient, pre- Cold; (2) Cryopreservation of neural stem cells: collect the cells in a centrifuge tube, count, add pre-cooled freezing solution, resuspend, draw the cell suspension into the cryopreservation tube, and then perform the program in the programmed cooling device Cooling; (3) After the program cooling is completed, transfer it to a liquid nitrogen tank for storage;

[0083] The precooling temperature of the step (1) is 4°C; the precooling temperature time is 50min;

[0084] The volume ratio of the medium to the nutrient is 1:0.9; the volume of the protective agent is 7% of the volume of the cold storage solution;

[0085] The protective agent includes DMSO and g...

Embodiment 2

[0097] Embodiment 2 of the present invention specifically provides a method for programmed cooling of a working cell bank of human neural stem cells, and its specific implementation method is the same as that of Embodiment 1, except that L-glutamine is not included.

[0098] Before cryopreservation: Before the last centrifugation and after resuspension in the freezing solution, take 100 μl of the cell suspension and put it on the Countstar cell fluorescence analyzer, and calculate the cell number and survival rate by the machine;

[0099] The operation steps of cell recovery are as follows: recover the cells on the 3rd day after freezing. When recovering, first absorb 20ml of complete medium into a 50ml centrifuge tube, and then put the frozen cells in a water bath at 38°C. After the cells are lysed, add to the corresponding centrifuge tube to resuspend. After balancing, centrifuge at 400g for 5min, discard the supernatant, add 20ml of complete medium to the pellet and mix well...

Embodiment 3

[0103] Embodiment 3 of the present invention specifically provides a method for programmed cooling of a working cell bank of human neural stem cells. The specific implementation method is the same as that of Embodiment 1, except that no trehalose is present.

[0104] Before cryopreservation: Before the last centrifugation and after resuspension in the freezing solution, take 100 μl of the cell suspension and put it on the Countstar cell fluorescence analyzer, and calculate the cell number and survival rate by the machine;

[0105] The operation steps of cell recovery are as follows: recover the cells on the 3rd day after freezing. When recovering, first absorb 20ml of complete medium into a 50ml centrifuge tube, and then put the frozen cells in a water bath at 38°C. After the cells are lysed, add to the corresponding centrifuge tube to resuspend. After balancing, centrifuge at 400g for 5min, discard the supernatant, add 20ml of complete medium to the pellet and mix well.

[01...

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Abstract

The invention relates to a programmed cooling method for cells, in particular to a programmed cooling method for a human neural stem cell working cell bank. The programmed cooling method of the human neural stem cell working cell bank comprises the following steps of: (1) preparing a cryopreservation solution, namely adding a protective agent into a centrifuge tube, shaking a culture medium to achieve uniform mixing, adding a nutritional agent, and performing pre-cooling; (2) performing cryopreservation of neural stem cells,namely, collecting the cells in the centrifuge tube, performing counting, adding a pre-cooled cryopreservation solution, performing resuspending, sucking a cell suspension into a cryopreservation tube, and performing programmed cooling in a programmed cooling instrument; and (3) after finishing the programmed cooling, transferring the cell suspension into a liquid nitrogen tank for storing the suspension. According to the method of the invention, under the condition of programmed cooling, the proper cryopreservation solution is selected, the survival rate of cells under cryopreservation is not influenced when the temperature during cell resuscitation is within a relatively large range, the activity of the cells is higher after the cells are cryopreserved and resuscitated, and the survival rate of the cells is more than 95%.

Description

technical field [0001] The invention relates to a method for programmed cooling of cells, in particular to a method for programmed cooling of a working cell bank of human neural stem cells. Background technique [0002] Neural stem cells are a type of cell population with proliferation, self-renewal ability and multi-directional differentiation potential, which can differentiate into various types of nerve tissue cells such as neurons, astrocytes and oligodendrocytes. Neural stem cells can be used to Treats damage and degeneration of brain tissue. The current science and technology can cultivate and expand neural stem cells in vitro, which can be used in life science research, drug screening test, clinical application research and other fields. Therefore, the establishment of a neural stem cell working library makes it possible to preserve neural stem cells for a long time, which can avoid the possibility of microbial contamination, genotype changes, and increased cell pass...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 朱灏
Owner 华夏源细胞工程集团股份有限公司
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