Ultra-low temperature preservation solution for animal cells and preserving method

A technology of cryopreservation and animal cells, applied in the field of biological preservation, to achieve the effect of simple operation and great practical value

Inactive Publication Date: 2009-07-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no literature report on the application of active ingredients of traditional Chinese medicine such as schisandrin, anisodamine, glycyrrhizic acid, danshensu, astragalus polysaccharide, and saikosaponin to cryopreservation of isolated cells

Method used

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  • Ultra-low temperature preservation solution for animal cells and preserving method
  • Ultra-low temperature preservation solution for animal cells and preserving method
  • Ultra-low temperature preservation solution for animal cells and preserving method

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Freshly isolated hepatocytes, Williams'E medium (added with 5mg / L matrine, 5mg / L anisodamine, 10mg / L schisandrin B, 10mg / L glycyrrhizic acid, 10mg / L danshensu, 10mg / L astragalus polysaccharide, 10 mg / L saikosaponin), 37° C., 5% CO2 incubator suspension pre-cultivation for 0.5 hours. Replace the medium with 1640 medium, the solution composition is 50% 1640 medium, add 10% dimethyl sulfoxide (DMSO) by volume, and also add 100mg / L matrine in the composite preservation solution , 50mg / L anisodamine, 50mg / L schisandrin, 50mg / L glycyrrhizic acid, 100mg / L astragalus polysaccharide, 100mg / L saikosaponin, etc., the final cell density is 1×10 7. Store overnight in a -80°C refrigerator (15-20 hours), and finally store in liquid nitrogen for 15 days. Thaw in a 37°C water bath, and then rewarm. The Williams’E medium used in the rewarming was added with the same protective agent as that used in the pre-cultivation, rewarmed and adhered to the wall for 24 hours, 48 ​​hours, and 96 ...

Embodiment 2

[0039] Chondrocytes harvested under sterile conditions were placed at 37°C for gel embedding and injected into hollow fiber filaments with a cell density of 1×10 5 . Put the hollow fiber assembly into a 5cm glass dish and immerse it in the DMEM / F12 medium (additional ingredients in the culture medium: 10mg / L matrine, 50mg / L anisodamine, 10mg / L Schizandrin, 10mg / L Glycyrrhizic acid, 10mg / L Danshensu, 10mg / L astragalus polysaccharide, 10mg / L saikosaponin, 3mMCa 2+ . ), 37°C, 5% CO 2 The incubator was left to pre-cultivate for 2 hours. Replace the medium with a protective agent and add HTS solution with the same content and composition, store overnight in a -80°C refrigerator (15-20 hours), and finally store in liquid nitrogen for 2 months. It was thawed in a water bath at 37°C, and the DMEM / F12 medium used for rewarming was also added with the same protective agent as that used for pre-cultivation. After rewarming and adherent culture for 24 hours, 48 ​​hours, and 96 hours, ...

Embodiment 3

[0043] The fibroblast cells harvested under sterile conditions were placed at 37°C for polysphere culture, injected into hollow fiber filaments, and the cell density was 1×10 6 . Put the hollow fiber into the silica gel tube, add RPMI 1640 medium (additional ingredients in the culture medium: 10mg / L matrine, 50mg / L anisodamine, 10mg / L schisandrin, 10mg / L glycyrrhizic acid, 10mg / L Danshensu, 10mg / L astragalus polysaccharide, 10mg / L saikosaponin, 3mMCa 2+ . ), 37°C, 5% CO 2 The incubator was pre-incubated for 1 h. Replace the medium with a protective agent and add UW solution with the same content and components, store overnight in a -80°C refrigerator (15-20 hours), and finally store in liquid nitrogen for 5 months. It was thawed in a water bath at 37°C, and the RPMI1640 medium used for rewarming was also added with the same protective agent as that used for pre-cultivation. After rewarming and adherent culture for 24 hours, 48 ​​hours, and 96 hours, various indicators wer...

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Abstract

The invention discloses a ultra-low temperature conserving solution of animal cell and conserving method thereof for conserving cell especially fresh separated primary animal cell, e.g. liver cell. Culturing fresh separated animal cell at 37 DEG C 0-24 h, changing culture medium as ultra-low temperature conserving solution at 0-4 DEG C then conserving 0-48 months at -80--196 DEG C. After conserving finished, recoverying culturing in a fresh cell culture medium at 37 DEG C, finally culturing in a normal animal cell culture medium. Adding Chinese medicine effective constituent and other chemical protective agent in conserving process and before and after culture medium to improve survival rate of animal cell after conserving at ultra-low temperature and function of cell. The ultra-low temperature conserving technique of separated animal cell is used for conveying and conserving biotype artifical organs in consist of biology artifical liver and other organ cell, also suit for application of animal cell in other medicine field.

Description

technical field [0001] The invention relates to biological preservation technology, in particular to a cryopreservation solution and preservation method for animal cells. Background technique [0002] At an ultra-low temperature below -80°C, the biochemical reactions inside various cells are extremely slow or even terminated. Using this principle, the cells are placed below -196°C so that their life activities are "fixed" rather than dead. The method of thawing in an appropriate way and restoring its activity is the cryopreservation of cells. [0003] There are many situations that cause cell damage or death during cryopreservation, and the damage to cells mainly includes the following aspects: 1) The fluidity of the cell membrane decreases, and the rigidity of the cell membrane will lead to further damage to the cells; 2) During the rewarming process Generate oxygen free radicals; 3) lead to cell necrosis and apoptosis. [0004] But so far, except for a few types of cells...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06A01N1/02
Inventor 孟琴鲁燕华沈冲
Owner ZHEJIANG UNIV
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