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Preparation method and application of NK cytokine mixture

A technology of NK cells and cytokines, which is applied in the field of preparation of NK cytokine mixtures, and can solve the problems of cumbersome cell culture steps

Inactive Publication Date: 2014-07-16
湖北华赛生物医药技术有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Traditional NK cell culture methods need to use PHA and / or tumor cell lines (such as K562, HFWT and K562-MB15-41BBL) as stimulators and trophoblasts to cultivate NK cells, which has cumbersome cell culture steps, using PHA and tumor cells Strains also pose safety concerns

Method used

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  • Preparation method and application of NK cytokine mixture

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Embodiment Construction

[0020] 1. In vitro culture, expansion and activation of NK cells

[0021] 1) Under sterile conditions, draw 20ml of peripheral blood from the patient's vein into a 50ml centrifuge tube, and add 300IU heparin sodium anticoagulant;

[0022] 2) Add physiological saline or PBS to dilute at 1:1;

[0023] 3) Add 5ml of lymphocyte separation medium to an empty 15ml centrifuge tube, take 10ml of diluted blood and slowly add it to a 15ml centrifuge tube, centrifuge at 1500rpm for 20min, take buffy coat cells and wash with normal saline, centrifuge at 1200rpm for 10min, then resuspend in PBS , repeat once, discard the supernatant;

[0024] 4) Resuspend the above cells with NK cell medium, and add anti-CD3 antibody (Orthoclone OKT-3; Ortho Biotech, Raritan, NJ), and adjust the final concentration of antibody to 10ng / ml;

[0025] NK cell culture medium Manufacturer Serum-free stem cell growth medium SCGM CellGenix, Germany 5% human serum Biowhittaker-Cambrex, US...

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Abstract

The invention belongs to the field of tumor biotherapy, and relates to a preparation method and an application of a NK cells-cytokines mixture prepared by using an activation method for NK cells. NK cells obtained according to the invention are tumor-killing cells which are cultured in vitro, activated and amplified and has a cytotoxic effect, and can directly kill tumor cells and enhance immunity after being fed back to the bodies of patients; NK cell culture supernatant prepared according to the invention and freeze-dried powder preparations prepared by freeze-drying and concentrating the NK cell culture supernatant contain a series of natural cytokine mixtures with bioactivity, and the mixture of natural cytokines has good antitumor activity in anti-tumor treatment.

Description

[0001] technical field [0002] The invention belongs to the field of tumor biotherapy, and relates to a preparation method and application of NK cytokine mixture. Background technique [0003] Natural killer cells (Natural killer cells, NK) are one of the first innate immune defense members in the human body, and they are also the strongest and most effective immune lymphocytes against cancer cells or viruses. Compared with cytotoxic T cells (CTL) or dendritic cells (Dendritic cells, DC), NK cells have a broad spectrum, are not limited by histocompatibility antigens, and can directly Launches an attack on cancer cells, hence the name natural killer cells. NK cells play the role of immune surveillance on a variety of infectious microorganisms and tumor cells, and have anti-tumor, anti-infection and immune regulation functions. Their target cells mainly include tumor cells, virus-infected cells, and some self-tissue cells (such as blood cells) ), parasites, etc. [0004] C...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/26A61P35/00A61K35/17
Inventor 吴皖王雄伟汤威邢文彦罗琳
Owner 湖北华赛生物医药技术有限公司
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