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a cell freezing medium

A cryopreservation 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 reducing the number of ice crystals, improving cell viability and avoiding pollution.

Active Publication Date: 2018-04-03
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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  • a cell freezing medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 Cryopreservation of human stem cells ADSCs

[0045]Mix dextran-40 solution and glucose injection evenly to obtain a dextran solution with a volume fraction of 1% dextran; add dimethyl sulfoxide, human serum albumin and dextran solution with a volume fraction of 20% to a PBS buffer solution to obtain a cell Freezing solution, the cell freezing solution contains 10% dimethyl sulfoxide, 5% human serum albumin, 2% dextran and 2% glucose. Store at 4°C.

[0046] According to the literature: Li Yanqi, Wang Hong improved the method of isolation and culture of umbilical cord-derived mesenchymal stem cells. "Chinese Tissue Engineering Research", 2014, 18(10): 1609-1614 The method in human stem cell ADSCs primary cells, after subculture 1. Obtain the cells of P3-P5.

[0047] 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 calculat...

Embodiment 2

[0052] Embodiment 2 Cryopreservation of human stem cells UC-MSC

[0053] 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.

[0054] 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.

[0055] Put the cryopreservation tube containing the cell suspension into a freezer bo...

Embodiment 3

[0059] The cryopreservation of embodiment 3 people's peripheral blood mononuclear cells (PBMC)

[0060] 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;

[0061] 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.

[0062] 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 cell cryopreservation fluid.The cell cryopreservation fluid is prepared from, by volume, 9-13% of dimethyl sulfoxide, 2-7% of human serum albumin, 1-3% of dextran, 1-5% of glucose and the balance solvent.Human serum albumin replaces animal serum to serve as a cell nutrient source, the pollution of animal source viruses is avoided, and nutrient substances of multiple components are effectively provided and can be directly absorbed and used by cells; in addition, a large amount of high-polymer protein is contained in human serum albumin and can form a hydration film, the number of crystals formed in the east village process is decreased, and mechanical loss and death of cells are reduced; by means of dextran, permeation pressure can be well maintained, the good permeation pressure environment can still be kept in the temperature decreasing process, and the cell death caused by temperature decreasing and permeation pressure changing is avoided, and the survival rate of cells recovering after being cryopreserved for a long time is increased.

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 Patents(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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