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Efficient culture method of CIK (cytokine induced killer) cells

A culture method and cell culture technology, which is applied in the field of high-efficiency CIK cell culture, can solve the problems of relatively large impact on the patient's immune system, affect the quality of life of the patient, and insensitivity to radiotherapy and chemotherapy, and improve the number of cells, killing activity, and proliferation speed Fast, high lethality effect

Inactive Publication Date: 2014-01-22
青岛麦迪赛斯生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Surgery, chemotherapy and radiotherapy are routine methods for tumor treatment. However, many cancer patients lose the opportunity to undergo surgery due to untimely detection. Chemotherapy and radiotherapy have a relatively large impact on the immune system of patients, seriously affecting the quality of life of patients. In addition, many cancer types are not sensitive to chemotherapy and radiotherapy

Method used

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  • Efficient culture method of CIK (cytokine induced killer) cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1) Coating of the culture flask: Take 100 micrograms of laminin and fibronectin respectively, dissolve them fully in 20 ml of phosphate buffer, filter the liquid with a 0.22 micron sterile filter membrane, and add it to the bottom with an area of ​​175 square centimeter culture flask at room temperature for 4 hours.

[0037] 2) Cell inoculation: Collect 50 ml of venous peripheral blood, slowly add it to the centrifuge tubes that have been added with 15 ml of lymphocyte separation medium, 25 ml per tube. Centrifuge at 2000 rpm for 15 minutes, take the white PBMC layer in the middle, wash it twice with normal saline, resuspend with 60 ml of serum-free medium, sample and count, the number of cells is 4.8×10 7indivual. Add cell suspension and inoculate into the culture flask without coating solution, add 60000 units of IFN-gamma, 60000 units of IL-2, 1200ng of anti-CD3 monoclonal antibody and 1200ng of anti-CD28 monoclonal antibody. The components of the serum-free medium...

experiment example 2

[0066] 1) Coating of the culture flask: Take 80 micrograms of laminin and fibronectin respectively, fully dissolve them in 20 ml of phosphate buffer, filter the liquid with a 0.22 micron sterile filter membrane, and add it to the bottom to reach an area of ​​175 square centimeter culture flask at room temperature for 4 hours.

[0067] 2) Cell inoculation: Collect 50 ml of venous peripheral blood, slowly add it to the centrifuge tubes that have been added with 15 ml of lymphocyte separation medium, 25 ml per tube. Centrifuge at 2000 rpm for 15 minutes, take the white PBMC layer in the middle, wash it twice with normal saline, resuspend with 60 ml of serum-free medium, sample and count, the number of cells is 4.2×10 7 indivual. Add the cell suspension to inoculate the culture flask without the coating solution, add serum-free medium to 70 ml, then add 70000 units of IFN-gamma, 70000 units of IL-2, 1400 ng of anti-CD3 monoclonal antibody and 1400 ng of anti-CD28 monoclonal anti...

experiment example 3

[0072] 1) Coating of the culture flask: Take 80 micrograms of laminin and fibronectin respectively, fully dissolve them in 20 ml of phosphate buffer, filter the liquid with a 0.22 micron sterile filter membrane, and add it to the bottom to reach an area of ​​175 square centimeter culture flask at room temperature for 4 hours.

[0073] 2) Cell inoculation: Collect 50 ml of venous peripheral blood, slowly add it to the centrifuge tubes that have been added with 15 ml of lymphocyte separation medium, 25 ml per tube. Centrifuge at 2000 rpm for 15 minutes, take the white PBMC layer in the middle, wash it twice with normal saline, resuspend with 60 ml of serum-free medium, sample and count, the number of cells is 5.3×10 7 indivual. Add the cell suspension to inoculate the culture flask without the coating solution, add serum-free medium to 80 ml, then add 80000 units of IFN-gamma, 80000 units of IL-2, 1600 ng of anti-CD3 monoclonal antibody and 1600 ng of anti-CD28 monoclonal anti...

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Abstract

The invention aims to provide an efficient culture method of CIK cells. The prepared CIK cell has the characteristics of high multiplication speed, large cell quantity, high cell viability, high killing capacity on cancer cells and the like. The efficient culture method mainly characterized in that a culture flask coated with laminin and fibronectin is used for culturing PBMCs (peripheral blood mononuclear cells), and the cells can be in a half-adherence state by means of the absorption effect of the two kinds of protein, accordingly, the cells can be stimulated by various factors, and the two kinds of protein can promote CIK cell multiplication; and with the adoption of an anti-CD28 monoclonal antibody, the co-stimulation signal of a T cell subset in the CIK cells can be stimulated, and the activation of the CIK cells is promoted. So far, a research report on jointly using laminin, fibronectin and the anti-CD28 monoclonal antibody to prepare the CIK cells is absent. The method for increasing the cell number and improving the killing activity by adding the three factors during preparation of the CIK cells is firstly provided.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to the cultivation of a cytokine-induced killer cell (Cytokine Induced killer, CIK), that is, the combination of various cytokines is used to stimulate peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cell, PBMC) Obtain a large number of killer cells with high killing activity. Background technique [0002] Malignant tumors are still one of the unsolved diseases in the world, and have become the leading cause of death in China. There are more than 2.8 million new cancer patients in my country every year. Surgery, chemotherapy and radiotherapy are routine methods for tumor treatment. However, many cancer patients lose the opportunity to undergo surgery due to untimely detection. Chemotherapy and radiotherapy have a relatively large impact on the immune system of patients, seriously affecting the quality of life of patients. In addition, many cancer types are n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0786
Inventor 徐矫健孙威
Owner 青岛麦迪赛斯生物科技有限公司
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