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Cell cryopreservation solution

A cryopreservation solution and cell technology, applied in the field of biomedicine, can solve the problems of restricting the clinical application of cryopreserved cells and low cell recovery rate, and achieve the effects of ensuring cell viability, improving cell viability and reducing ice crystal formation.

Active Publication Date: 2016-06-22
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cell recovery rate after long-term cryopreservation is low, only about 85%, which directly restricts the clinical application of cryopreserved cells.

Method used

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  • Cell cryopreservation solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Cryopreservation of human stem cells ADSCs

[0040] Add DMSO and 20% human serum albumin to the Lonza basal medium respectively to form a cell freezing solution containing 10% DMSO and 5% human serum albumin.

[0041] According to the literature: Tian Lin, Sun Xiaofang, Liu Haibo. Isolation, culture and biological characteristics of human adipose stem cells. "Chinese Tissue Engineering Research", 2012, 16(32): 5946-5952 to obtain ADSCS primary cells, after subculture , to obtain cells from P3 to P5.

[0042] After resuspending with PBS, centrifuge at 1500r / min for 5min, discard the supernatant. Resuspend the cells with the cell freezing medium at 4°C, take 50 μL of the cell suspension, and calculate the cell viability and number after mixing according to cell cliff (v):0.4% trypan blue (v)=1:1; adjust Cell density to a cryopreservation density of 1×10 6 cells / mL, divide the cell suspension into cryopreservation tubes, 1.5mL / tube; mark and freeze 6 tubes....

Embodiment 2

[0047] Embodiment 2 Cryopreservation of human stem cells UC-MSC

[0048] According to the literature: Li Yanqi, Wang Hongyi. Improvement of the method for the isolation and culture of human umbilical cord-derived mesenchymal stem cells. "Chinese Tissue Engineering Research", 2014, 18(10): 1609-1614 to obtain the original UC-MSC Generation cells, after subculture, obtained cells from P3 to P5.

[0049] After resuspending with PBS, centrifuge at 1500r / min for 5min, discard the supernatant. Use the cell cryopreservation solution prepared in Example 1 at 4°C to resuspend the cells, take 50uL of the cell suspension, mix according to cell cliff (v): 0.4% trypan blue (v) = 1:1, and perform cell Calculation of viability and quantity; adjust the cell density to a frozen storage density of 1×10 6 cells / mL, divide the cell suspension into cryopreservation tubes, 1.5mL / tube; mark and freeze 6 tubes.

[0050] Put the cryopreservation tube containing the cell suspension into a freezer bo...

Embodiment 3

[0054] The cryopreservation of embodiment 3 people's peripheral blood mononuclear cells (PBMC)

[0055] Obtain human peripheral blood mononuclear cells (PBMC) according to the literature: Chen Dan, Wang Xiaodong. Separation of three methods for isolating human peripheral blood mononuclear cells. "Journal of Tianjin Medical University", 2014.6:483-485;

[0056] After resuspending with PBS, centrifuge at 1500r / min for 5min, discard the supernatant. Use the cell cryopreservation solution prepared in Example 1 at 4°C to resuspend the cells, take 50uL of the cell suspension, mix according to cell cliff (v): 0.4% trypan blue (v) = 1:1, and perform cell Calculation of viability and quantity; adjust the cell density to a frozen storage density of 3×10 6 cells / mL, divide the cell suspension into cryopreservation tubes, 1.5mL / tube; mark and freeze 6 tubes.

[0057] Put the cryopreservation tube containing the cell suspension into a freezer box, put it in a -80°C refrigerator, and tran...

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Abstract

The invention provides a cell cryopreservation solution prepared from components in percentage by volume as follows: 9%-13% of dimethyl sulfoxide, 2%-7% of human serum albumin and 82%-87% of a culture medium, wherein the culture medium comprises a Lonza basal culture medium and / or a DMEN high-glucose culture medium. The culture medium and the human serum albumin jointly constitute a nutritional system with rich and multiple components, stable and direct nutrition supply is provided for cells, and the cell survival rate is guaranteed. Besides, in the temperature decrease process, macromolecular substances such as protein and the like form hydrated shells, ice crystal formation is reduced, and mechanical damage and death of the cells due to ice crystal formation when the temperature decreases are avoided, so that the cell viability after long-term cryopreservation recovery is increased. Experimental results indicate that the cell recovery rate is 94%-98%, 94%-97% and 92%-94% respectively after the cryopreservation solution is used for cryopreserving the cells for 1 month, 6 months and 12 months.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular to a cell cryopreservation solution. Background technique [0002] In the process of subculture and daily maintenance of cell culture, a lot of expense is required in terms of culture equipment, culture medium and various preparations, and once the cells leave the living body to start primary culture, its various biological characteristics will gradually change and become With the increase of the number of passages and the change of the environmental conditions in vitro, there are new changes. Therefore, timely cell cryopreservation is very necessary. [0003] At present, the most commonly used technology for cell cryopreservation is liquid nitrogen cryopreservation, which mainly adopts the slow freezing method of adding an appropriate amount of protective agent to cryopreserve cells, such as using glycerol or dimethyl sulfoxide (DMSO) as protective agent, because if Cells are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 陈海佳王一飞葛啸虎卢瑞珊
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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