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CIK cells with high cytotoxic activity for immune treatment of tumor cells

An immunotherapy and tumor cell technology, applied in the field of cellular immunity, can solve problems such as impaired immune function, weakened immune cell function, and affecting the curative effect of CIK cells

Active Publication Date: 2015-12-23
天津市津华生物科技有限公司
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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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  • CIK cells with high cytotoxic activity for immune treatment of tumor cells
  • CIK cells with high cytotoxic activity for immune treatment of tumor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Induced preparation and detection of CIK cells

[0019] 1. Induction and preparation of CIK cells

[0020] (1) Take 30 mL of peripheral blood from the patient and add an equal volume of RPMI1640 culture medium (Invitrogen Company), and mix well.

[0021] (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%.

[0022] (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

[0048] Example 2: Induced preparation of CIK cells

[0049] (1) Take 30 mL of peripheral blood from the patient and add an equal volume of RPMI1640 culture medium (Invitrogen Company), and mix well.

[0050] (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%.

[0051] (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.

[0052] (4) Add complete medium, count the cells, adjust the mononuclear ...

Embodiment 3

[0059] Example 3: Induced preparation and detection of CIK cells

[0060] (1) Take 30 mL of peripheral blood from the patient and add an equal volume of RPMI1640 culture medium (Invitrogen Company), and mix well.

[0061] (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) at 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%.

[0062] (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.

[0063] (4) Add complete medium, count the cells, adjust the mono...

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Abstract

The invention discloses CIK cells with high cytotoxic activity for immune treatment of tumor cells. The CIK cells are prepared through the following steps that firstly, peripheral blood of a patient is sampled, and the peripheral blood mononuclear cell part is collected; secondly, the peripheral blood mononuclear cell part collected in the first step is centrifuged, supernate is discarded, and mononuclear cells are obtained; thirdly, the mononuclear cells are taken according to 1*106 / mL, and a complete medium including 500-2,000 IU / mL of gamma interferon and 80-120 ng / mL of a compound is added, and induction culturing is started, wherein the complete medium includes RPMI 1640 cell culture fluid with 10% of FBS by the volume percent; fourthly, after the cells in the third step are cultured for 20-28 hours, 50-1,000 ng / mL anti-human CD3 monoclonal antibodies and 500-2,000 IU / mL of recombination human IL-2 are added, induction culturing is continued, subculture is conducted once every other two to three days, the induction culturing time is 13 to 21 days, and the CIK cells are obtained. The CIK cells can be used for immune treatment of the tumor cells.

Description

technical field [0001] The invention belongs to the field of cellular immunity, and in particular relates to a CIK cell with high cytotoxic activity for tumor cell immunotherapy and a method for preparing the CIK cell 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 immu...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
Inventor 杨廷稳
Owner 天津市津华生物科技有限公司
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