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Method for preparing cytokine-induced killer cells

A cell and nuclear cell technology, which is applied in the field of preparing CIK cells, can solve the problems of decreased killing activity of CIK cells, and achieve the effects of improving killing activity, quality of life, and survival period

Inactive Publication Date: 2012-12-19
英普乐孚生物技术(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by the present invention is the problem that the killing activity of CIK cells decreases while the method for preparing CIK cells in the prior art greatly expands CIK cells, and then provides a method that can ensure the number of CIK cells and Method for preparing CIK cells capable of improving its killing activity

Method used

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  • Method for preparing cytokine-induced killer cells

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Experimental program
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Effect test

Embodiment 1

[0019] Mononuclear cells were collected and isolated from peripheral blood, separated by density gradient of human lymphocyte separation medium; mononuclear cells were taken from the interface layer and placed in a medium suitable for lymphocytes. In this example, AIM-V serum-free medium was selected. , adjust the cell concentration to 0.5×10 6 / ml, add CD3 monoclonal antibody, IL-2, IFN-γ, adjust CD3 monoclonal antibody concentration to 30ng / ml, IL-2 concentration to 100u / ml, IFN-γ concentration to 500u / ml, and culture for 12 days; Add IFN-α and IL-2 to the above culture medium, adjust the concentration of IFN-α to 100u / ml, and the concentration of IL-2 to 100u / ml, and continue to culture for 2 days to obtain CIK cells.

Embodiment 2

[0021] Mononuclear cells were collected and isolated from peripheral blood, separated by density gradient of human lymphocyte separation medium; mononuclear cells were taken from the interface layer and placed in a medium suitable for lymphocytes. In this example, AIM-V serum-free medium was selected. , adjust the cell concentration to 1.5×10 6 / ml, add CD3 monoclonal antibody, IL-2, IFN-γ, adjust the concentration of CD3 monoclonal antibody to 70ng / ml, the concentration of IL-2 to 500u / ml, and the concentration of IFN-γ to 1500u / ml, and cultivate for 8 days; Add IFN-α and IL-2 to the above culture medium, adjust the concentration of IFN-α to 1000u / ml, and the concentration of IL-2 to 500u / ml, and continue to culture for 6 days to obtain CIK cells.

Embodiment 3

[0023] Mononuclear cells were collected and isolated from peripheral blood, separated by density gradient of human lymphocyte separation medium; mononuclear cells were taken from the interface layer and placed in a medium suitable for lymphocytes. In this example, AIM-V serum-free medium was selected. , adjust the cell concentration to 1×10 6 / ml, add CD3 monoclonal antibody, IL-2, IFN-γ, adjust the concentration of CD3 monoclonal antibody to 50ng / ml, the concentration of IL-2 to 300u / ml, and the concentration of IFN-γ to 1000u / ml, and cultivate for 10 days; Add IFN-α and IL-2 to the above culture medium, adjust the concentration of IFN-α to 500u / ml, and the concentration of IL-2 to 300u / ml, and continue to culture for 4 days to obtain CIK cells.

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Abstract

The invention discloses a method for preparing cytokine-induced killer (CIK) cells. The method comprises the following steps: 1) collecting and separating individual karyocytes from peripheral blood; 2) placing the individual karyocytes obtained in the step 1) in a culture medium suitable for lymphocytes, removing monocytes by the adherence characteristic of the monocytes, and adding CD3 monoclonal antibodies, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in the rest to culture the lymphocytes to CIK cells; and 3) culturing the culture solution obtained in the step 2) for 8-15 days, and then adding interferon-alpha (IFN-alpha) and IL-2 to culture continuously. Compared with the cells which are cultured without adding IFN-alpha, the cells cultured by the method have higher killing property to tumor cells which is increased by 50%-200%. Through the clinical treatments of more than 100 malignant tumor patients, the response rate which is related to the progressive disease (PR), the complete response (CR), the magnetic resonance imaging (MR) and the stable disease (SD) can reach 75%, wherein the response rate (PR+CR+MR) can reach 50%. By adopting the method for preparing CIK cells, the life cycle of advanced tumor patients can be prolonged and the life quality of the patients can be increased.

Description

technical field [0001] The invention relates to a method for preparing CIK cells, belonging to the field of biotechnology. Background technique [0002] Tumor biological therapy is the fourth treatment mode after surgery, radiotherapy and chemotherapy. CIK cells, namely cytokine-induced killer cells (Cytokine-Induced killer, CIK) is a new type of immunocompetent cells, CIK has strong proliferation ability, strong cytotoxicity, and certain immune characteristics. One of the most effective cells for tumor biotherapy. CIK cells have stronger cell proliferation and stronger Anti-tumor cell effect, and less toxic side effects. [0003] The efficacy of CIK cells in treating tumors mainly depends on the number and killing activity of the infused cells. The current traditional CIK cell preparation method is to first isolate the mononuclear cells in the peripheral blood, use the culture medium suitable for lymphocytes, add appropriate amount of CD3 monoclonal antibody, IL-2, IFN-...

Claims

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Application Information

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IPC IPC(8): C12N5/078
Inventor 高岱清兰克涛马伟魏晓芳赵鹏解西河李长优孙伟红
Owner 英普乐孚生物技术(上海)有限公司
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