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Culture method of autologous peripheral blood lymphocyte DC-CIK (Dendritic Cell- Cytokine-induced Killer)

A technology of DC-CIK and lymphocytes, which is applied in the field of in vitro culture of immune cells, can solve the problems of increased expression and does not take into account the effect of T regulatory cell content on tumor immune escape, so as to achieve the effect of improving killing activity

Inactive Publication Date: 2015-02-18
ADLAI NORTYE BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Insufficiency of the current DC-CIK technology: In the microenvironment of the tumor, the expression of CTLA4 and PD-1 on the surface of CIK cells increases, which binds to the corresponding ligands on DCs, thereby mediating the negative immune regulation mechanism and inhibiting CIK cell killing function
Existing DC-CIK techniques do not take into account the effect of T regulatory cell content on tumor immune escape

Method used

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  • Culture method of autologous peripheral blood lymphocyte DC-CIK (Dendritic Cell- Cytokine-induced Killer)
  • Culture method of autologous peripheral blood lymphocyte DC-CIK (Dendritic Cell- Cytokine-induced Killer)
  • Culture method of autologous peripheral blood lymphocyte DC-CIK (Dendritic Cell- Cytokine-induced Killer)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Extraction of Peripheral Blood Mononuclear Cells

[0088] (1) Collect 30-100mL of peripheral blood with a sodium citrate anticoagulant tube, transfer the peripheral blood to a 50mL centrifuge tube, centrifuge at 2000rmp for 15min;

[0089] (2) Discard the upper layer of plasma, dilute the blood cells with normal saline so that the ratio of blood cells: normal saline is 1:1, and mix well;

[0090] (3) Pipette 15mL of Ficoll lymphocyte separation solution into a 50mL centrifuge tube, and add the mixed cell suspension evenly and slowly to the upper layer to form a complete interface;

[0091] (4) 1600rpm, centrifuge for 20min, obvious stratification can be seen;

[0092] (5) Collect interface mononuclear cells and wash them twice with PBS;

[0093] (6) Resuspend the cells in PBS and count them for later use.

Embodiment 2

[0094] Example 2 Preparation of DC cells

[0095] (1) Use X-VIVO15 serum-free medium to adjust the cell concentration of the isolated mononuclear cells to 1*10 6 Cells / mL were inoculated into T75 culture flasks, adhered to the wall for 2 hours, and the suspended cells were separated into new T175 culture flasks;

[0096] (2) Add 15mL of X-VIVO15 serum-free medium to the adherent cells, and add rhGM-CSF1000U / mL and rhIL-4 500U / mL, at 37°C, 7.5%CO 2 Cultivated in an incubator;

[0097] (3) On the third day, X-VIVO15 serum-free medium was replaced by half, and rhGM-CSF and rhIL-4 were added to keep the concentration unchanged;

[0098] (4) Add 50ug / mL of tumor antigen protein extracted from the human kidney cancer cell line ACHN on the 5th day, add rhTNF-a 500U / mL on the 6th day, and continue culturing until the 7th day.

Embodiment 3

[0099] Example 3 Preparation of CIK cells

[0100] (1) Place the suspended cells separated in Step 1 of Example 2 at 37°C, 7.5% CO 2 incubator cultivation;

[0101] (2) On the third day, add X-VIVO15 serum-free medium to 100mL, and add 1000U / mL IL-2;

[0102] (3) On the fourth day, add X-VIVO15 serum-free medium to 240mL, and add IL-2 to keep the concentration constant.

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Abstract

The invention relates to a culture method of autologous peripheral blood lymphocyte DC-CIK. The culture method comprises the following steps: (1) separating mononuclear cells of peripheral blood; (2) conducting isolated culture of DC cells; (3) conducting induced culture of CIK cells; (4) co-culture the DC and CIK cells to prepare DC-CIK cells, and adding 100 ng / mL of CTLA-4 (Cytotoxic Lymphocyte Antigen) mAb and 100 ng / mL of PD-1 (Programmed Cell Death-1) mAb; (5) centrifuging and collecting the cultured DC-CIK cells. The culture method disclosed by the invention is to add a plurality of monoclonal antibodies and cell factors in a blood serum medium to increase the activation efficiency and the amplification efficiency of the effector cell colony, in particular load the CTLA4 antibodies and the PD-1 antibodies in vitro to cover the CTLA4 and PD-1 molecules on the surface of all the CIK cells, and enable the molecules not to be bound with corresponding receptors on the DC, so as to effectively reduce the content of T regulatory cells, and further improve the lethal effect of the CIK cells on tumors.

Description

technical field [0001] The invention belongs to the technical field of in vitro culture of immune cells, in particular to a method for culturing autologous peripheral blood lymphocyte DC-CIK. Background technique [0002] Adoptive immunotherapy is to infuse sensitized lymphocytes (with specific immunity) or their products to people with low cellular immunity (such as tumor patients) to obtain anti-tumor immunity. It is an immune system used to treat tumors. therapy. [0003] Dendritic cells (Dendritic cells) are the most powerful antigen presenting cells (Antigen presenting cell, APC) in the body. They are the only APCs that can stimulate the maturation of initial T lymphocytes, and can present antigens to CD8+ T lymphocytes. Thereby exerting the killing effect of T lymphocytes. [0004] Cytokine induced killer cells (CIK) are human peripheral blood mononuclear cells co-cultured with various cytokines (such as anti-CD3 monoclonal antibody, IL-2 and IFN-γ, etc.) for a perio...

Claims

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Application Information

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IPC IPC(8): C12N5/0784C12N5/0783
Inventor 何学松何南海叶晓峰林晓峰杨东晖路杨
Owner ADLAI NORTYE BIOPHARMA CO LTD
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