A kind of immune cell serum-free cryopreservation liquid and cryopreservation method

A technology of immune cells and cryopreservation solution, which is applied in the field of cell culture, can solve the problems of changing cell thermodynamics, chemical and physical environment, single cell damage, biological damage, etc., and achieve the goal of improving survival rate, good proliferation ability and good activity Effect

Active Publication Date: 2021-04-06
湖南旭智生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the cryopreservation process, the existing cryopreservation solution will inevitably cause damage to individual cells, and even toxicity, especially during the cryopreservation process, the thermodynamic, chemical and physical environment of the cells will be significantly changed, accompanied by biological damage danger

Method used

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  • A kind of immune cell serum-free cryopreservation liquid and cryopreservation method

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0025] The preparation process of NK cells in Examples 1 to 3 is as follows:

[0026] (1) Take an appropriate amount of blood, divide it evenly into two 50ml centrifuge tubes, centrifuge at 650g at room temperature (both reduce the lifting speed to 1), and centrifuge for 15min;

[0027] (2) Remove the upper layer of plasma after centrifugation, add an equal volume of PBS to the lower layer of cells and mix to obtain a cell suspension;

[0028] (3) Carefully add to the Ficoll layer to keep the layers clear, room temperature, and centrifuge;

[0029] (4) Aspirate the peripheral blood mononuclear cell (PBMC) layer, aspirate the cell layer at the junction of the two liquid surfaces as much as possible, add PBS to mix, centrifuge, and wash the cells again in the same way to obtain peripheral blood mononuclear cells (PBMC);

[0030] (5) The above-mentioned PBMC cells were screened with magnetic beads to obtain NK cells.

[0031] The present invention has no special limitation on t...

Embodiment 1

[0033] A serum-free cryopreservation solution for NK cells, including serum-free RPMI 1640 medium and a cell cryopreservation agent, the volume fraction of the cell cryopreservation agent in the serum-free cryopreservation solution for immune cells is: 8%. Based on the final volume of the stock solution, the content of each component in the cell protection agent is: polyethylene glycol 4.0 mg / mL, nonylphenol polyoxyethylene ether-10 2.0 mg / mL, saponins 8.5 μg / mL, Sodium carboxymethyl cellulose 10.5 μg / mL, polyglycerol fatty acid ester 16.0 μg / mL, bamboo leaf flavone 38.0 μg / mL.

[0034] A method for freezing NK cells. The NK cells to be frozen are mixed with the above-mentioned serum-free cell freezing solution to obtain a cell suspension, and the concentration of cells in the cell suspension is 5×10 6 cells / mL, the above cell suspension was placed in a sterile cryopreservation tube and cooled to -80°C, frozen for 12 hours, and then frozen in liquid nitrogen.

Embodiment 2

[0036] A serum-free cryopreservation solution for NK cells, including serum-free RPMI 1640 medium and a cell cryopreservation protection agent, the volume fraction of the cell cryopreservation protection agent in the serum-free cryopreservation solution for immune cells is: 5%. Based on the final volume of the stock solution, the content of each component in the cell protection agent is: polyethylene glycol 3.5 mg / mL, nonylphenol ethoxylate-10 1.5 mg / mL, saponins polysaccharide 8.5 μg / mL, Sodium carboxymethyl cellulose 9.0 μg / mL, polyglycerol fatty acid ester 15.0 μg / mL, bamboo leaf flavone 35.0 μg / mL.

[0037] A method for freezing NK cells. The NK cells to be frozen are mixed with the above-mentioned serum-free cell freezing solution to obtain a cell suspension. The concentration of cells in the cell suspension is 9×10 6 cells / mL, the above cell suspension was placed in a sterile cryopreservation tube and cooled to -80°C, frozen for 20 hours, and then frozen in liquid nitrog...

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Abstract

The invention discloses a serum-free cryopreservation solution for immune cells, which is characterized in that it comprises a basal medium and a cell cryopreservation protective agent, and the cell cryopreservation protective agent is composed of the following raw materials: polyethylene glycol, nonylphenol poly Oxyethylene ether-10, acacia polysaccharide, sodium carboxymethylcellulose, polyglyceryl fatty acid ester, bamboo leaf flavone. In the present invention, polyethylene glycol and nonylphenol polyoxyethylene ether-10 are used to replace DMSO to change the permeability of the cell membrane and at the same time protect the cell membrane, so that the free water in the cell can be excluded from the cell and reduce the risk of cryopreservation. The formation of ice crystals in the cells during the process damages the cells. Acacia polysaccharides, sodium carboxymethylcellulose, and polyglycerol fatty acid esters are added as osmotic pressure stabilizers to maintain a stable osmotic pressure in the extracellular environment. Added with bamboo leaf flavonoids, it helps cells recover quickly after recovery. The invention also provides a cryopreservation method for immune cells, which is simple and convenient, and meets the demands for immune cells in scientific research, medical treatment and other fields.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a serum-free cryopreservation solution and a cryopreservation method for immune cells. Background technique [0002] Immune cells refer to cells involved in or related to immune responses, including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Immune cells can be divided into many types, and various immune cells play important roles in the human body. Immune cells include phagocytes and lymphocytes, and T cells and B cells in lymphocytes are the main cell groups that exert specific immunity. Mononuclear cells isolated from blood include: T cells, B cells, NK cells and DC cells, etc., which are the initial cell source in immune cell therapy. After induction and expansion of various cytokines, a group of cells with high killing activity can be obtained. immune cell population. [0003] As an effective method to preserve cells, cell cryopreservat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 张晓盈王小奇苏琳琳黄睿龙孙向东
Owner 湖南旭智生物科技有限公司
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