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An efficient in-vitro amplification culture method for natural killer cells having high killing capability

A culture method and in vitro expansion technology, which is applied in the field of high-efficiency in vitro expansion and culture of natural killer cells, to achieve the effects of less damage to the body, easy collection of materials, and a wide range of sources of materials

Inactive Publication Date: 2017-08-29
溯源生命科技股份有限公司
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Problems solved by technology

At present, the challenges faced by NK cell immunotherapy include: how to enhance the recognition and infiltration of NK cells expanded in vitro, and how to improve the survival rate of NK cells in vivo

Method used

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Embodiment Construction

[0014] This specific embodiment adopts the following technical scheme: the high-efficiency in vitro expansion and culture method of natural killer cells with high lethality is as follows: Step 1: configure the culture medium, add 5mL plasma to every 100mL MEM-a medium, add IL- 210U / mL, IL-15 100U / mL, IL-21 10U / mL, and SCF 50ng / mL, coat the culture flask, dilute the fibronectin to 0.1mg / mL with PBS or normal saline, draw 5mL into 10cm culture After coating in a dish overnight at 4°C, suck out the residual liquid and set aside;

[0015] Step 2: Sampling: Take 5mL-10mL of venous blood or umbilical cord blood, with a viability ≥90%, for the preparation of mononuclear cells, take 5mL-10mL of lymphocyte separation medium, carefully transfer the venous blood to the separation medium, and operate gently to avoid Break the junctional liquid level, centrifuge at 1800r / min for 15min, absorb the upper layer, namely the plasma layer, for later cell culture, carefully absorb the middle buff...

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Abstract

An efficient in-vitro amplification culture method for natural killer cells having high killing capability is disclosed and belongs to the technical field of cell culture. The method includes (1) preparing a medium; (2) sampling; (3) centrifuging mononuclear cells with 400 g of double-antibody PBS having a concentration of 500 U / mL for 10 min, fully rinsing the mononuclear cells for three times, and inoculating the mononuclear cells to a culture dish; (4) in the second day to the sixth day, observing the color of the medium and counting, and supplementing the fresh medium every 2-3 days; (5) after the seventh day, reducing the autologous plasma content by 1%; (6) purifying the NK cells; and (7) cryopreserving the purified CD56<+>NK cells in liquid nitrogen by utilizing a serum-free cryopreserving liquid. The method has advantages of easily available raw materials, capability of preparing a high number of NK cells having activity from a small amount of peripheral blood or umbilical blood, suitability for large-scale culture, low damage to bodies, and the like. The sources of the materials are wide, and in-vitro storage quantity is high. The prepared natural killer cells are suitable for autologous treatment and free of immunogenicity.

Description

Technical field: [0001] The invention relates to a high-efficiency in vitro expansion and culture method for natural killer cells with high lethality, and belongs to the technical field of cell culture. Background technique: [0002] Comprehensive treatment is an important treatment principle for malignant tumors. In addition to traditional chemotherapy, radiotherapy and surgery, the status of immunotherapy is increasing day by day. Since the 1980s, scientists discovered that lymphokine-activated autologous killer cells (Lymphokine Activated Killer, LAK) can treat malignant tumors after in vitro amplification of large doses of IL-2, and thus proposed the concept of adoptive immune cell therapy for tumors . In the adoptive therapy of tumor immunity, NK cells are important effector cells. NK cells have been widely studied because they have cytotoxicity without activation and have a wide anti-tumor spectrum. In vitro experiments have confirmed that NK cells have a killing effe...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/125C12N2501/2302C12N2501/2315C12N2501/2321C12N2533/52
Inventor 汪晓敏袁卫平张孝兵周丽英万谦
Owner 溯源生命科技股份有限公司
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