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Biological agent for effectively killing and wounding tumor cells

A technology of biological preparations and tumor cells, applied in animal cells, antineoplastic drugs, vertebrate cells, etc., can solve problems such as insufficient purity and slow cell growth

Active Publication Date: 2015-12-02
BEIJING BIOHEALTHCARE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biological preparation of the present invention has the characteristics of strong tumoricidal activity, broad tumoricidal spectrum, non-MHC restriction, etc., which fundamentally solves the problem of insufficient purity of NK cells after culture, and also solves the problem of insufficient purity between cells during the whole lymphocyte culture process. Competing nutrients from the original proportion of the smaller cells lead to slow growth problems

Method used

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  • Biological agent for effectively killing and wounding tumor cells
  • Biological agent for effectively killing and wounding tumor cells
  • Biological agent for effectively killing and wounding tumor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1PBMC separation

[0029] 1) Centrifuge 50-80ml of peripheral blood collected intravenously with heparin anticoagulant blood collection tubes at 1500r / mim for 5min, carefully absorb the upper serum with a pipette, and resuspend the cell pellet with an equal amount of PBS;

[0030] 2) Slowly add an equal volume of Ficoll (dextran-diatrizoate meglumine) (purchased from General Electric Medical Group, item number: 17-5442-02 / 03) to the separation medium of the above cell suspension, and set the natural lifting speed of the centrifuge , centrifuged at 2000r / min for 20min, and after centrifugation, absorb the middle buffy coat cells as peripheral blood mononuclear cells;

[0031] 3) Wash the above mononuclear cells twice with PBS, and remove the Ficoll separation solution.

Embodiment 2

[0032] Example 2 Cell Sorting

[0033] 1) Add fluorescently-labeled flow sorting antibodies CD56-PE (BD Company, Cat. No.: 555516) and CD19-FITC (BD Company, Cat. No.: 555412) with fluorescent labels to the cells obtained in Example 1, and keep away from light at room temperature Incubate for 20 minutes;

[0034] 2) Wash off excess antibody with PBS;

[0035] 3) Cell sorting was carried out with the flow cytometry sorter Infiux of BD Company in the United States, wherein CD56+ was used as the standard for NK cell sorting, CD19+ was used as the standard for sorting mature B lymphocytes, and CD56-CD19- was used as the standard for sorting CIK cells;

[0036] 4) Harvest and sort the NK cells, mature B lymphocytes and CIK cells as the basic cells for culture.

Embodiment 3

[0037] Example 3 Cell culture (NK cell culture, mature B cell culture, T cell culture)

[0038] 1) The CIK cells, mature B lymphocytes, and NK cells obtained in Example 2 were transferred into the 75cm 2 In the cell culture flask, add 10ml each of the different media required for culturing the three types of cells, and store at 37°C in 5% CO 2 Cultivated in a carbon dioxide incubator, the composition of the special medium for different cells is as follows:

[0039] 2) Composition of special medium

[0040] 3)

[0041] 4) The above-mentioned cells were supplemented with the above-mentioned medium in time according to the growth of the cells during the culture process, and the same amount of MICA (Reliatech Company, product number: 101-M569) protein and MICB (Reliatech Company, product number: 101-M570) protein at a concentration of 500 ng / ml in the culture medium, and at the same time add Lenalidomide (Medchemexpress Company, product number: HY-A0003) stimulator with a c...

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Abstract

The invention provides a biological agent for effectively killing and wounding tumor cells. Human NK (natural killer) cells, mature B lymphocyte cells and CIK (cytokine-induced killer) cells are amplified through induction in vitro, and are combined according to a certain proportion, and the biological agent for effectively killing and wounding the tumor cells is provided. The biological agent with a combined tumor killing effect has the advantages of high tumor killing activity, wide tumor killing spectra and zero MHC (major histocompatibility complex) limitation.

Description

technical field [0001] The invention relates to the field of in vitro culture of immune cells, in particular to a biological preparation for killing tumor cells, a preparation method and an application. Background technique [0002] Tumor-infiltrating lymphocytes kill tumor cells through two mechanisms: one is that lymphocytes and tumor cells contact each other, directly secrete cytotoxic granules and perforin to bind with tumor target cells, and lyse tumor cells; the other is to secrete cells with cytotoxic function. Factors, such as tumor necrosis factor, interferon-mediated indirect killing. [0003] Cytokines-induced killer cells (CIK) are obtained by co-cultivating human peripheral blood mononuclear cells with various cytokines (such as anti-CD3 monoclonal antibody, IL-2 and IFN-γ, etc.) for a period of time in vitro a group of heterogeneous cells. Because this kind of cell expresses CD3+ and CD56+ two kinds of membrane protein molecules at the same time, it is also c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61P35/00C12N5/0781C12N5/0783
Inventor 卢戌杨照敏刘静维
Owner BEIJING BIOHEALTHCARE BIOTECH
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