Preparing method of human DCCIK immunocompetent cell

An immune activity and cell technology, applied in the biological field, can solve the problems of cumbersome operation and complicated technology, and achieve the effect of simplifying operation steps, easy technology and shortening time.

Inactive Publication Date: 2014-10-01
SHANGHAI YUYAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problem of cumbersome operation and complex technology in the existing DCCIK culture technology, DC cells need to be induced to mature from adherent monocytes and then mixed with CIK cells, the present invention provides a simple, efficient and easy Popularized and applied DCCIK preparation technology

Method used

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  • Preparing method of human DCCIK immunocompetent cell
  • Preparing method of human DCCIK immunocompetent cell
  • Preparing method of human DCCIK immunocompetent cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 , the preparation of DCCIK cells and CIK cells

[0026] 1.1, Preparation of DCCIK cells of the present invention

[0027] The preparation method of DCCIK cell of the present invention is as follows:

[0028] Peripheral blood was collected from patients, and mononuclear cells were separated by density gradient centrifugation of human lymphocyte separation medium. Mononuclear cells were inoculated into GT-T551 serum-free medium (purchased from Japan Takara Company), and the cell concentration was adjusted to 1×10 6 / ml, add IFN-γ (1000U / ml), IL-2 (500U / ml), anti-CD3 monoclonal antibody (20ng / ml), IL-4 (1000U / ml), GM-CSF (500U / ml). Then at 37℃, 5%CO 2 Under the condition of culture, immature DC cells can be induced after 5 days, and TNFα (500U / ml) is added to make DC mature. After 7 days, add GT-T551 serum-free medium containing IL-2 (500 U / ml) and subculture every other day for 7-10 days to obtain DCCIK cells.

[0029] 1.2. Preparation of DCCIK cells in th...

Embodiment 2

[0035] Example 2 , cell number, cell phenotype and tumor cell killing activity

[0036] The three kinds of cells prepared in Example 1 were tested for cell number, cell phenotype and tumor cell killing activity, and the results are as follows.

[0037] 2.1. Determination of cell number

[0038] The three kinds of cells were cultured for different time, and the cell expansion fold was determined by cell counting, and the results are shown in Table 1.

[0039] Table 1. Cell expansion multiple results

[0040]

[0041]

[0042] As shown in Table 1, the results show that the multiplication factor of DCCIK cultured by the mixed induction culture of the present invention is basically the same as that of DCCIK cells obtained by the method of the prior art, and the multiplication factor of CIK cell culture is increased by more than 4 times when cultured for 15 days.

[0043] 2.2. Cell phenotype analysis

[0044] The three kinds of cells cultured for 15 days were applied wit...

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Abstract

A preparing method of a human DCCIK immunocompetent cell is disclosed. The method includes steps of: (1) separating a mononuclear cell from human peripheral blood; (2) inoculating the mononuclear cell to a culture medium suitable for lymphocyte culturing, adding IFN[gamma], IL-2, an anti-CD3 monoclonal antibody, GM-CSF and IL-4, and culturing for 3-5 days; (3) adding TNF[alpha], and continuously culturing for 1-2 days; and (4) adding IL-2, and continuously culturing for 7-10 days to obtain the DCCIK cell. A composition of various cytokines and stimulating factors is added directly into the mononuclear cell so that the DC cell and lymphocyte induce at the same time and mutually stimulate maturity, thus simplifying operation steps, reducing experiment consumable items and shortening cell culturing time. In addition, normalized experiment process technology can be easily mastered, thus facilitating clinic popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of human DCCIK immunocompetent cells. Background technique [0002] A large number of basic and clinical studies at home and abroad have proved that the combination of immunotherapy and traditional surgery, radiotherapy, and chemotherapy plays an important role in controlling the disease, improving the quality of life and prolonging the survival of tumor patients. Recent studies have found that non-specific immune cells can Kill tumor stem cells and prevent tumor recurrence and metastasis (R. Tallerico et al, The Journal of Immunology, 2013, 190(5): 2381–90). At present, the most widely used non-specific immune cell therapy methods at home and abroad are CIK and DCCIK cell therapy. [0003] CIK cells, Cytokine Induced Killer (Cytokine Induced Killer), are immune effector cells induced from human peripheral blood mononuclear cells in vitro by a variet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078A61K35/14A61P35/00
Inventor 徐荻张尚泉吴涤梵
Owner SHANGHAI YUYAN BIOTECH CO LTD
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