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Animal derived serum-free cell freezing medium

A technology of animal source and cryopreservation solution, applied in the field of cell therapy, can solve the problems of increasing animal pathogens, pollution, virus transmission, etc., and achieve the effects of low cost, reducing animal pathogen pollution, and simple preparation methods

Inactive Publication Date: 2016-09-21
SHANGHAI ANGECON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, the fetal bovine serum used in cell cryopreservation increases the possibility of animal pathogen contamination. For example, mad cow disease has occurred in many European countries, and its serum contains the virus that causes mad cow disease. The use of frozen cells will lead to the spread of such viruses
In addition, studies have shown that cells that have been in contact with fetal bovine serum for a long time will endocytose fetal bovine serum in the solution medium, and stem cells after endocytosis of fetal bovine serum may undergo some protein expression changes. The immune reaction caused by xenogeneic animal protein cannot be directly used for clinical infusion

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0017] Cell culture (taking cell CIK as an example)

[0018] Use lymphocyte separation medium density gradient centrifugation to separate peripheral blood mononuclear cells, or to revive frozen mononuclear cells. Centrifuge at 500 g of normal saline for 5 minutes, and remove the supernatant. Count the cells and make 1*106 / ml cell suspension with the corresponding culture medium. Add 2ml of culture medium and cell suspension into a 6-well plate, with 3 parallel wells in each group. On the first day, 1000IU / ml of INF-r was added and cultured in a carbon dioxide incubator. After 24 hours, 50ng / ml of CD3 monoclonal antibody and 500IU / ml of IL-2 were added. Afterwards, every 48 hours, add corresponding culture medium and 500IU / ml IL-2, and count to 1*106 / ml cell concentration. The cell morphology was observed after co-cultivation for 14 days, and the phenotype and viability of CIK cells were detected at 14 days. The specific test results are shown in Table 1.

Embodiment 2

[0020] According to the volume ratio, 5% HSA was mixed with 85% HES and 10% DMSO to prepare cell cryopreservation solution 1, which was stored at 4°C for later use.

[0021] The CIK cells expanded in Example 1 were treated with a concentration of 5×10 7 The concentration per ml was suspended in the above-mentioned cryopreservation solution 1, placed in a programmed cooling box and placed in a -80°C ultra-low temperature refrigerator overnight, and transferred to liquid nitrogen for cryopreservation the next day.

[0022] After 1 month, 3 months, 6 months and 1 year after cryopreservation, the cells were recovered in a 37°C water bath. After 5 days of culture, the cell phenotype and viability were detected. The specific test results are shown in Table 1.

Embodiment 3

[0024] Mix 10% HSA with 80% HES and 10% DMSO according to the volume ratio to prepare cell cryopreservation solution 1, and store it at 4°C for later use.

[0025] The CIK cells expanded in Example 1 were treated with a concentration of 5×10 7 The concentration per ml was suspended in the above-mentioned cryopreservation solution 2, placed in a programmed cooling box and placed in a -80°C ultra-low temperature refrigerator overnight, and transferred to liquid nitrogen for cryopreservation the next day.

[0026] After 1 month, 3 months, 6 months and 1 year after cryopreservation, the cells were recovered in a 37°C water bath. After 5 days of culture, the cell phenotype and viability were detected. The specific test results are shown in Table 1.

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Abstract

The invention belongs to the field of cell therapy, and especially relates to an animal derived serum-free cell freezing medium. The animal derived serum-free cell freezing medium comprises, by volume, 5-15% of human albumin, 5-15% of dimethyl sulfoxide and 70-90% of hydroxyethyl starch. The cell freezing medium contains no animal derived serum, so no exogenous proteins are introduced, and the animal pathogen pollution possibility is reduced; and the animal derived serum-free cell freezing medium is safe and effective in additional culture or clinic direct transfusion after cryopreservation resuscitation, and also has the advantages of simple preparation method, low cost and good clinic application prospect. The activity of recovered cells reaches about 90%, and the cell phenotype basically has no change, so the physiologic functions and the biological functions of the recovered cells are kept. The animal derived serum-free cell freezing medium is suitable for refrigerated storage of mononuclear cells and other various cells.

Description

technical field [0001] The invention belongs to the field of cell therapy, in particular to a cell cryopreservation solution without animal source serum and a preparation method thereof. Background technique [0002] Cell cryopreservation is one of the methods for long-term preservation of cells. Cryopreservation of donor cells can maintain their viability and function, so that they can be revived for use when needed. It is of great significance to both clinical and basic research. The main factors affecting the activity of cryopreservation and recovery of cells include the method of freezing and recovery and the choice of cryogen. The process of cell cryopreservation will significantly change the thermodynamic, chemical and physical environment of cells, with the risk of causing biological damage. In order to minimize cell damage during cryopreservation and recovery, chemical and temperature manipulations must be further optimized. Cells are frozen at -70°C to -80°C, and...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 苗嘉奕卫培培王晓明
Owner SHANGHAI ANGECON BIOTECH
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