High-cytotoxic activity CIK cells and application in tumor cellular immunotherapy

A technology for immunotherapy and tumor cells, which is applied in the direction of cell culture active agents, animal cells, anti-tumor drugs, etc., and can solve problems such as impaired immune function, weakened immune cell function, and low ability to kill tumors

Inactive Publication Date: 2016-09-28
丁露露
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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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  • High-cytotoxic activity CIK cells and application in tumor cellular immunotherapy
  • High-cytotoxic activity CIK cells and application in tumor cellular immunotherapy
  • High-cytotoxic activity CIK cells and application in tumor cellular immunotherapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Induced preparation and detection of CIK cells

[0018] 1. Induction and preparation of CIK cells

[0019] (1) Take 30 mL of peripheral blood from the patient and add an equal volume of RPMI 1640 culture medium (Invitrogen Company), and mix well.

[0020] (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%.

[0021] (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

[0046] Example 2: Induced preparation of CIK cells

[0047] (1) Take 30 mL of peripheral blood from the patient and add an equal volume of RPMI 1640 culture medium (Invitrogen Company), and mix well.

[0048] (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%.

[0049] (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.

[0050] (4) Add complete medium, count the cells, adjust the mononuclear...

Embodiment 3

[0057] Example 3: Induced preparation and detection of CIK cells

[0058] (1) Take 30 mL of peripheral blood from the patient and add an equal volume of RPMI 1640 culture medium (Invitrogen Company), and mix well.

[0059] (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%.

[0060] (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.

[0061] (4) Add complete medium, count the cells, adjust t...

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Abstract

The invention discloses a CIK cell with high cytotoxic activity and its application in tumor cell immunotherapy. The CIK cell is prepared by the following steps: (1) taking the peripheral blood of a patient, and collecting the peripheral blood mononuclear cell part; The peripheral blood mononuclear cells collected in step (1) were partially centrifuged, and the supernatant was discarded to obtain mononuclear cells; 6 /mL cells, add the complete medium containing 500~2000IU/mL gamma interferon and 80~120ng/mL compound (I), and start the induction culture; the complete medium includes RPMI 1640 cell culture medium containing 10% FBS by volume percentage; (4) After 20-28 hours of cell culture in step (3), add 50-1000ng/mL anti-human CD3 monoclonal antibody and 500-2000IU/mL recombinant human IL-2, and continue to induce culture, every 2-3 days The CIK cells were subcultured once, and the induced culture time was 13-21 days to obtain CIK cells. The CIK cells can be used for tumor cell immunotherapy.

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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCC12N5/0646A61K35/17C12N2501/2302C12N2501/24C12N2501/515C12N2501/999
Inventor 丁露露
Owner 丁露露
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