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A CIK cell and a preparation method and application thereof

A cell and nuclear cell technology, applied in the field of CIK cells and their preparation, can solve the problems of limited CIK cell activity maintenance time, limited time to exert effect, and many intermediate liquid replacement operations, so as to reduce the number of liquid replacement operations and survival time Effects of Prolongation, Type and Reduction of Working Concentration

Active Publication Date: 2018-12-07
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, the culture process of CIK cells requires a variety of cytokines and antibodies, and the cost is high
The culture time is also relatively long, and there are many intermediate liquid replacement operations, which are prone to pollution and other situations.
In addition, affected by the activation-induced apoptosis mechanism, the activity maintenance time of CIK cells is greatly limited, which limits the time for them to exert their effects in vivo

Method used

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  • A CIK cell and a preparation method and application thereof
  • A CIK cell and a preparation method and application thereof
  • A CIK cell and a preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Technical process for the preparation of CIK cells of the present invention

[0036] (1) Isolation and culture of PBMC from tumor patients

[0037] Collect 5 mL of peripheral blood from malignant tumor patients after surgery or chemotherapy, separate PBMCs with human lymphocyte separation medium, and culture the cells in GT-T551 medium containing 10% fetal bovine serum, 100 units / mL penicillin and 100 μg / mL streptomycin ( Takara).

[0038] (2) Induce CIK cells

[0039] After PBMC cells were treated with 25ng / mL IL-7 for 48 hours, 1 μg / mL antiCD3 and 1 μg / mL antiCD28 were added at the same time, stimulated at a low concentration for 48 hours, and then 1 μg / mL antiCD95 was added to continue culture for 4 days.

[0040] CIK cells were obtained without changing medium in the middle.

Embodiment 2

[0041] Example 2: Detection of proliferation and activation effects of CIK cells

[0042] (1) IL-7 and antiCD95 synergistically stimulate the proliferation of PBMC cells in tumor patients

[0043] The PBMCs used in the cell proliferation experiment were labeled with CFSE, and the PBMCs were cultured in the dark. The CIK cells were prepared as described above. The control cells were stimulated with 1 μg / ml of antiCD3 and antiCD28 at low concentrations and then added with IgG control antibody. Cells were collected, loaded for flow cytometry detection, and the Proliferation function of Flowjo software was used to analyze the percentage of cell proliferation.

[0044] (2) IL-7 and antiCD95 synergistically stimulate T lymphocyte subsets and phenotype detection in PBMC of tumor patients.

[0045] CIK cells and controls were prepared in the same way as described above. After the cells were collected, they were stained with CD4, CD8, and CD45RA flow cytometry antibodies, and then flo...

Embodiment 3

[0047] Example 3: IL-7 and CD95 molecules co-stimulate the tumor killing activity detection of PBMC of tumor patients

[0048] 1. In vitro cell killing experiment of tumor cell lines

[0049] The MKN-45 gastric cancer-derived cell line was cultured in RMPI1640 medium containing 10% fetal bovine serum, 100 units / ml penicillin and 100 μg / ml streptomycin. When MKN-45 cells were cultured to the logarithmic growth phase, they were seeded in 96-well plates at a density of 1×10^4 per well and cultured overnight. CIK cells and controls were prepared as above. After 4 days, PBMC were collected and MKN-45 cells were plated overnight to synchronize.

[0050] PBMCs and MKN-45 were counted, and different numbers of PBMCs were added to the wells of the MKN-45 cell culture plate replaced with serum-free medium, so that the ratio of the number of PBMCs to MKN-45 (effector to target ratio, E / T ratio) is 0.5:1, 1:1, 2:1, 4:1, 16:1 and other gradients.

[0051] In order to avoid the impact of ...

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Abstract

A CIK cell and a preparation method and application thereof are disclosed, belonging to the technical field of biology. According to a technical scheme adopted, the CIK cell is prepared by firstly pre-stimulating a peripheral blood mononuclear cell with IL-7, then stimulating the cell with low-concentration anti-CD3 and anti-CD28, and finally adding anti-CD95 for stimulation to prepare the CIK cell. Compared with the prior art, the variety of added cytokines / antibodies and working concentrations of the method are lower than those of other methods, thus saving the cost. The CIK cell prepared bythe method has more obvious tumor killing effect in an in-vitro anti-tumor experiment than cells prepared by present methods, and the method does not need liquid changing during in-vitro culture, issimple to operate and can still maintain cell activity better than that of cells prepared by other methods. The CIK cell has a wide prospect in the field of controlling and relieving progression of malignant tumor or other diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CIK cell and its preparation method and application. Background technique [0002] CIK (Cytokine-induced killer cells, cytokine-induced killer cells) is a mixed cell population of T cells and NK cells (natural killer cells, natural killer cells). CIK is generally human PBMC (peripheral blood mononuclear cells, peripheral blood mononuclear cells) in vitro in IFN-γ (interferon-gamma), anti-CD3 antibody (anti-CD3), anti-CD28 antibody (anti-CD28), interleukin-1 (IL-1), interleukin-2 (IL-2) and other cytokines / antibody induction culture preparation. When T lymphocytes cannot recognize malignant cells lacking MHC markers, CIK cells can mediate MHC-restricted and non-restricted anti-tumor cytotoxicity, thereby mediating a more rapid immune response. These characteristics make CIK cells very promising in tumor immunotherapy. [0003] However, the culture process of CIK cells...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0638C12N5/0646C12N2501/2307C12N2501/999A61K2300/00
Inventor 尹悦马乐
Owner XI AN JIAOTONG UNIV
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