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Preparation method of cytotoxicity-enhanced efficient target killing NK/CIK (Natural Killer)/( Cytokine Induced Killer) cells

A cytotoxic and cellular technology, applied in the field of NK/CIK cell preparation, can solve the problems of poor NK cell proliferation ability, and achieve the effect of improving cell purity and effective biological activity, significantly targeting and killing tumors, and significantly proliferating ability.

Active Publication Date: 2015-12-23
丛秀丽
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  • Application Information

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Problems solved by technology

[0022] The technical problem to be solved by the present invention is to overcome the defects in the prior art that magnetic bead sorting is required for NK cell culture, the obtained NK cells have poor proliferative ability, and tumor cells insensitive to NK cells have no killing ability, and provide a kind of cell Preparation method of highly efficient targeted killing NK / CIK cells with enhanced toxicity

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  • Preparation method of cytotoxicity-enhanced efficient target killing NK/CIK (Natural Killer)/( Cytokine Induced Killer) cells
  • Preparation method of cytotoxicity-enhanced efficient target killing NK/CIK (Natural Killer)/( Cytokine Induced Killer) cells
  • Preparation method of cytotoxicity-enhanced efficient target killing NK/CIK (Natural Killer)/( Cytokine Induced Killer) cells

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Embodiment 1

[0059] A preparation method for highly efficient targeted killing of NK / CIK cells with enhanced cytotoxicity, comprising the following steps:

[0060] 1. Blood collection and mononuclear cell isolation

[0061] (1) Collect 50-80 mL of peripheral blood or umbilical cord blood, collect it in a blood collection bag containing heparin sodium anticoagulant, and shake it up quickly to avoid coagulation;

[0062] (2) Put the blood collection bag and the biological ice bag together into the incubator and keep the temperature at 4-8°C; transport them to the laboratory within 12 hours;

[0063] (3) Dilute the blood with an equal volume of normal saline and mix it evenly, and slowly superimpose it on the lymphocyte separation medium in the centrifuge tube, so that the volume ratio of the diluted blood to the lymphocyte liquid is 3:2, and pay attention to maintaining the boundary of the liquid layer clear;

[0064] (4) Centrifuge the centrifuge tube at 900g and 20°C for 25 minutes. Afte...

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Abstract

The invention discloses a preparation method of cytotoxicity-enhanced efficient target killing NK / CIK (Natural Killer) / ( Cytokine Induced Killer) cells. The preparation method is applicable to culture of the NK / CIK and DC (Dendritic Cell)-NK / CIK cells derived from peripheral blood and cord blood. The preparation method comprises the following steps: acquiring blood; separating mononuclear cells; culturing NK / CIK cells by adopting suspending cells; culturing DC cells by adopting adherent mononuclear cells; carrying out mixed culture on the NK / CIK cells and the DC cells according to a proportion of 10: 1. According to the preparation method disclosed by the invention, mononuclear cells which are not sorted are adopted for culture for 14 days by utilizing efficient cytokine combination to generate NK / CIK mixed effector cells in a system, and the purity of the cells is nearly 90%, wherein NK cells account for 52.4%, and CIK cells account for 33.3%, and cells expressing killing marks account for over 80%, and all the NK cells highly express activating marks CD161; the induced DC-NK / CIK cells have remarkable multiplication capability and target killing capability to tumors, and the killing capability is remarkably improved especially in aiming at tumor cells insensitive to the NK cells, and thus the range of the NK cells in clinical tumor therapies is expanded.

Description

technical field [0001] The invention relates to a preparation method of NK / CIK cells, in particular to a preparation method of highly efficient targeted killing NK / CIK cells with enhanced cytotoxicity. Background technique [0002] Mononuclear cells: Refers to white blood cells with a single nucleus obtained from blood or bone marrow separated by Ficoll. They are mainly divided into two groups, monocytes and lymphocytes. [0003] NK cells (natural killer, NK): are the main bearer of the body's natural immunity, almost participate in the regulation process of important links such as the occurrence, development and effect of the immune system, and are related to anti-tumor, anti-viral infection and immune regulation. NK cells are mainly distributed in peripheral blood, accounting for 5-10% of PBMC, NK activity is also found in lymph nodes and bone marrow, but the level is lower than that in peripheral blood. The killing activity of NK cells is not limited by MHC and does not...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/0784
Inventor 丛秀丽
Owner 丛秀丽
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