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Preparation method for CIK cells with high cytotoxic activity

A cytotoxic and cellular technology, applied in the field of cellular immunity, can solve the problems of impaired immune function, weakened immune cell function, and low ability to kill tumors, and achieve the effects of high proliferation, high purity, and high cytotoxic activity.

Active Publication Date: 2016-01-27
北京大地同年生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immune function of tumor patients is impaired, and the function of immune cells is weakened. Therefore, the ability of CIK cells prepared from peripheral blood of tumor patients to kill tumors is low, which affects the efficacy of CIK cells.

Method used

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  • Preparation method for CIK cells with high cytotoxic activity
  • Preparation method for CIK cells with high cytotoxic activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Induced preparation and detection of CIK cells

[0021] 1. Induction and preparation of CIK cells

[0022] (1) Take 30 mL of peripheral blood from the patient and add an equal volume of RPMI1640 culture medium (Invitrogen Company), and mix well.

[0023] (2) Add 60 mL of the doubly diluted peripheral blood obtained in step (1) to the interface of 30 mL of lymphocyte separation medium (Shanghai Huajing Biological High-tech Co., Ltd.) (specific gravity 1.077) (do not destroy the interface), and use 2000 rpm, centrifuge for 20 minutes, take the middle layer - the buffy coat layer rich in lymphocytes, and add 20 mL of complete medium. A small amount of cells were taken for cell counting and trypan blue live cell count staining, and the results showed that the live cells reached 99%.

[0024] (3) Centrifuge the buffy coat layer containing complete medium obtained in step (2) at 1000 rpm for 10 minutes, discard the supernatant, and precipitate mononuclear cells. ...

Embodiment 2

[0050] Example 2: Induced preparation of CIK cells

[0051] (1) Take 30 mL of peripheral blood from the patient and add an equal volume of RPMI1640 culture medium (Invitrogen Company), and mix well.

[0052] (2) Add 60 mL of the doubly diluted peripheral blood obtained in step (1) to the interface of 30 mL of lymphocyte separation medium (Shanghai Huajing Bio-Technology Co., Ltd.) (specific gravity 1.077) (do not destroy the interface), and use 1500 rpm, centrifuge for 20 minutes, take the middle layer - the buffy coat layer rich in lymphocytes, and add 20 mL of complete medium. A small amount of cells were taken for cell counting and trypan blue live cell count staining, and the results showed that the live cells reached 99%.

[0053] (3) Centrifuge the buffy coat layer containing complete medium obtained in step (2) at 1500 rpm for 7 minutes, discard the supernatant, and precipitate mononuclear cells.

[0054] (4) Add complete medium, count the cells, adjust the mononuclea...

Embodiment 3

[0061] Example 3: Induced preparation and detection of CIK cells

[0062] (1) Take 30 mL of peripheral blood from the patient and add an equal volume of RPMI1640 culture medium (Invitrogen Company), and mix well.

[0063] (2) Add 60 mL of the doubly diluted peripheral blood obtained in step (1) to the interface of 30 mL of lymphocyte separation medium (Shanghai Huajing Bio-Technology Co., Ltd.) (specific gravity 1.077) (do not destroy the interface), and use 2500 rpm, centrifuge for 15 minutes, take the middle layer - the buffy coat layer rich in lymphocytes, and add 20 mL of complete medium. A small amount of cells were taken for cell counting and trypan blue live cell count staining, and the results showed that the live cells reached 99%.

[0064] (3) Centrifuge the buffy coat layer containing complete medium obtained in step (2) at 1500 rpm for 7 minutes, discard the supernatant, and precipitate mononuclear cells.

[0065] (4) Add complete medium, count the cells, adjust ...

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Abstract

The invention discloses a preparation method for CIK cells with high cytotoxic activity. The preparation method comprises the following steps: (1) taking peripheral blood of a patient and collecting mononuclear cell parts of the peripheral blood; (2) centrifuging the mononuclear cell parts of the peripheral blood collected in the step (1), and removing the supernate and acquiring the mononuclear cells; (3) adding the mononuclear cells into a complete medium containing 500-2000IU / mLy interferon and 80-120ng / mL Turrapubin A according to the standard of 1*10<6> / mL cells, starting to induce and culture, wherein the complete medium contains RPMI 1640 cell culture fluid containing FBS with the volume percent at 10%; (4) after culturing the cells for 20-28 hours in the step (3), adding 50-1000ng / mL anti-human CD3 monoclonal antibody and 500-2000IU / mL recombination human IL-2, continuing inducing and culturing, wherein the subculture is performed once per 2-3 days and the time of the whole inducing culture is 13-21 days, thereby acquiring the CIK cells. According to the preparation method provided by the invention, the CIK cells with high purity, high proliferation ability and high cytotoxic activity can be acquired and applied to cellular immunotherapy for tumors.

Description

technical field [0001] The invention belongs to the field of cellular immunity, and in particular relates to a preparation method of CIK cells with high cytotoxic activity. Background technique [0002] Immunotherapy, as the fourth mode of comprehensive tumor treatment, has received more and more attention. Tumor immunotherapy mainly includes tumor vaccine therapy, cytokine therapy, adoptive cellular immunotherapy and monoclonal antibody immunotherapy, etc. Tumor vaccines use tumor cells or tumor antigens to induce the body's specific cellular and humoral immune responses, enhance the body's ability to fight cancer, and prevent tumor growth, spread, and recurrence. The cytokines used in anti-tumor research mainly include interferons, interleukins, colony-stimulating factors, etc. Among them, IFN-α, IL-2, and granulocyte-macrophage colony-stimulating factor are the most common. Adoptive cellular immunity includes lymphokine activated killer cells, natural killer cells, tumo...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
Inventor 熊桂荣
Owner 北京大地同年生物科技有限公司
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