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Preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate, associated CIK cells and application

A proliferative and cytotoxic technology, applied in the field of surgery, I, C, can solve the problems of low cell vitality, tumor killing ability, no significant increase in cell number, and limited tumor treatment effect, and is suitable for large-scale promotion and application. Excellent tumor killing efficiency, ingeniously designed effect

Inactive Publication Date: 2012-12-19
上海优立赛尔生物医药科技有限公司
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Problems solved by technology

CD3 in CIK cells thus obtained + , CD4 + , CD8 + The content of T cells is low, the cell viability and tumor killing ability are low, and the therapeutic effect on tumors is very limited
On the other hand, the statistics of the existing CIK cell preparation methods show that in about 10% of cases / times, CIK cells cannot be expanded from peripheral blood mononuclear cells, and the number of cells does not increase significantly during the expansion process

Method used

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  • Preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate, associated CIK cells and application
  • Preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate, associated CIK cells and application
  • Preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate, associated CIK cells and application

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preparation example Construction

[0036] The preparation method of the present invention uses a sorting method to remove CD4 before culturing + CD25 + Treg cells, add IFN-γ (1000U / ml) in the culture, and coat with anti-CD3 monoclonal antibody (1ug / ml) to increase the cytotoxic activity, while adding IL-6 (100ng / ml), PGE2 (10ng / ml) to inhibit Monocyte to CD4 + CD25 + Treg cells differentiate, add insulin to a final concentration of 1ug / ml, IL-2 (1000U / ml) promotes cell proliferation.

[0037] Therefore, one of the technical solutions adopted by the present invention to solve the above-mentioned technical problems is: a method of inhibiting the conversion of monocytes to CD4 + CD25 + The method of Treg cell differentiation is to add IL-6 (100ng / ml) and PGE2 (10ng / ml) to the culture medium.

[0038] The second technical solution adopted by the present invention to solve the above-mentioned technical problems is: a method for promoting the proliferation of CIK cells, that is, adding a cell culture solution containing in...

Embodiment 1

[0041] Example 1 Preparation of CIK cells

[0042] 1. Preparation of peripheral blood mononuclear cells (PBMC) (Ficoll density gradient method)

[0043] Use a sterile syringe to collect 30-50ml of patient's anticoagulant blood under aseptic conditions. After centrifugation at 1500rpm / min for 15 minutes, aspirate the upper plasma, inactivate at 56°C for 30 minutes, put it at 4°C, and centrifuge at 3000rpm / 8min after 30 minutes to remove precipitation Put it in the refrigerator at 4℃ for later use. Use normal saline to double-dilute the blood cell pellet, add the human lymphocyte separation solution with a specific gravity of 1.077 to the diluted blood in a centrifuge tube at a ratio of 1:2, centrifuge at 2000rpm / min for 20 minutes, carefully extract the white blood cell layer, and wash twice with normal saline , PBMC is obtained after low speed centrifugation.

[0044] 2. Mini MACS removes CD4 + CD25 + Treg cells (Mini MACS highly magnetic bead separation column (Germany MACS compan...

Embodiment 2

[0047] Example 2: Detection of CIK cells

[0048] 1. CIK cell live cell detection

[0049] After adding PHA and INF-γ, most of the cells are still in suspension; after 3 days, the cell volume increases, the cells gradually aggregate into clusters, the cells are transparent, and the cytoplasm is rich; starting from the 5th day, the cells begin to proliferate in large quantities and the cell morphology is diverse. Increased cell colonies; take 100ul of CIK cells on day 1, 5, 7, 9, 11, 13, 15, 17, and 19 of culture, add 100ul of 0.4% placental blue staining solution, live cells will not be stained, dead cells are stained Into blue. See figure 1 Shown (where the experimental group is the CIK cell prepared in the present invention, that is, the CIK cell prepared in Example 1, and the control group is the conventional CIK culture (according to the invention titled "lymphocyte culture medium and methods and applications for culturing lymphocytes"" Chinese invention For CIK cells disclos...

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Abstract

The invention relates to a preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate. The preparation method comprises the steps of: (1) sorting and removing CD4<+>CD25<+>Treg cells of peripheral blood mononuclear cell to obtain CIK pre-cells; (2) cultivating the CIK pre-cells in a cell culture fluid containing 100ng / ml of PHA, 100ng / ml of IL-6 and 10ng / ml of PGE2 for 24h; (3) transferring the CIK pre-cells to a cell culture bottle coated with 1microgramme / ml of CD3 monoclonal antibody, and adding 1000U / ml of IFN-gamma for cultivating for 48h; (4) adding 1000U / ml of IL-2 and 100ng / ml of IL-1alpha for cultivating for 4 days; and (5) adding 1microgramme / ml of insulin to continuously cultivate for 7-14 days. The invention further provides associated CIK cells, a method for inhibiting the peripheral blood mononuclear cell to be differentiated to the CD4<+>CD25<+>Treg cells and a method for promoting the proliferation of the CIK cells. The preparation method of the CIK cells provided by the invention is skillful in design, and the tumor killing cells-CIK cells prepared have stronger proliferation capacity, higher cytotoxic activity and better tumor killing efficiency, so that the clinical efficacy is improved, and the preparation method is appropriate for wide application in a large scale.

Description

Technical field [0001] The present invention relates to the cross-technology field of life sciences and medicine, in particular to the technical field of enhanced immune cells, that is, Cytokine-Induced Killer Cell (CIK), in particular to a high proliferation capacity and high cytotoxicity Active and high survival rate CIK cell preparation method, related CIK cells and application. Background technique [0002] As the threat of tumors to human survival and health is becoming more and more serious, the treatment model to deal with tumors is also undergoing rapid changes. New drugs, new technologies, and new methods for various tumor treatments emerge in an endless stream. Among them, the immune cell therapy of various tumors is very active. It has become an important development direction in tumor biotherapy. [0003] Adoptive Cellular Immunotherapy (ACI or AIT) for tumors refers to the transfer of immune cells (specific and non-specific) with anti-tumor activity to tumor patients ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0786A61K35/14A61P35/00A61P31/00A61P37/02A61K35/17
Inventor 凌丹彦戴书缙
Owner 上海优立赛尔生物医药科技有限公司
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