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
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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
[0038] 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
[0041] 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 composition, 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 RPMI 1640 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 w...
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