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Peripheral blood NK cell in-vitro efficient expansion method

An NK cell and in vitro expansion technology, applied in the field of cell culture, can solve the problems of hidden safety hazards, complicated operation, high cost, etc., and achieve the effect of enhancing killing ability and meeting clinical needs

Active Publication Date: 2017-08-22
青岛麦迪赛斯医疗技术有限公司
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Problems solved by technology

Using feeder cells to culture NK cells can obtain a large number of high-purity NK cells, but the introduction of human cells has potential safety hazards; the method of using immunomagnetic beads can quickly obtain a small amount of NK cells, but it can only be used for NK cell research. Few are not suitable for clinical use, and the cost is high, and the operation is complicated; the existing method of cytokine combination to stimulate the expansion of NK cells in PBMC can only make the expansion of NK cells about 150 times, and the purity of NK cells is about 60%. And the quantity and purity cannot meet the therapeutic requirements

Method used

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Experimental program
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Effect test

Embodiment 1

[0034] 1) Coating of culture flasks: respectively, 25 micrograms of CD16 monoclonal antibody, 25 micrograms of CD56 monoclonal antibody, and 50 nanograms of CD3 monoclonal antibody were dissolved in 5 ml of PBS and fully dissolved, and added to a culture flask with a bottom area of ​​75 square centimeters, 4 ℃ overnight in the dark;

[0035] 2) Cell inoculation: a) Peripheral blood 40ml, whole blood centrifuged at 700g for 15min; b Autologous plasma preparation: take 21ml of upper plasma, inactivate at 56°C for 30min, let stand at 4°C for 15min, centrifuge at 900g for 15min, supernatant is autologous plasma Standby; c take about 5ml of the middle buffy coat (mononuclear cells) into 15ml of PBS, dilute and mix; d then slowly add the diluted monocytes to the surface of the lymphatic separation solution to make the gap between the buffy coat and the fence separation solution There are clear layers between them, the ratio of lymph separation liquid to mononuclear cell liquid is 3:...

Embodiment 2

[0043] In order to verify whether the combination of lower and higher concentrations of cytokines will affect the proliferation and purity of NK cells, this experiment set up two sets of examples, the first set of NK cell induction culture process in Example 2 CD3 monoclonal antibody, CD16 monoclonal antibody , the concentration of CD56 monoclonal antibody is different from Example 1, and the lower concentration in the combination is used in Example 2, wherein the final concentration of CD3 monoclonal antibody is 1ng / ml, the concentration of CD16 monoclonal antibody is the final 5μg / ml, and the final concentration of CD56 monoclonal antibody is 5μg / ml. The final concentration of antibody was 5 μg / ml. The CD3 monoclonal antibody, CD16 monoclonal antibody, and CD56 monoclonal antibody concentration in the NK cell induction culture process of the second group of Example 3 are also different from those in Example 1, and those used in Example 3 are higher concentrations in the combi...

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Abstract

The invention provides a peripheral blood NK cell in-vitro efficient expansion method. According to the peripheral blood NK in-vitro efficient expansion method, mononuclear cells (PBMC) are separated from peripheral blood through a lymph separation solution, the separated mononuclear cells are added to a CD16 monoclonal antibody, and a CD56 monoclonal antibody and a low-concentration CD3 monoclonal antibody are subjected to stimulation and co-culture in a culture bottle which is coated in advance, so that NK cells are preferentially expanded; then, IL-15 is used for activating the cultured cells on the fifth day, and culture continues to obtain a large number of high-purity NK cells. According to the method, the PBMCs do not need to be purified, a large number of high-purity NK cells can be obtained without using feeder cells, NK cells with the purity of 90% or above can be obtained after culture is conducted for 17 days, the cell expansion multiple of the cells is 400 times or above, requirements of clinical application can be met, the cost is reduced, steps are simple, and operability is high.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for cultivating peripheral blood NK cells (Natural killer cells) in vitro with high efficiency expansion. Background technique [0002] NK cells, natural killer cells, were discovered in 1975 and belong to non-phagocytic lymphocytes. The cytoplasm is rich in perforin and granzyme, and has the function of recognizing and lysing tumor cells. It is mainly derived from bone marrow CD34 + Lymphocytes mainly exist in peripheral blood, accounting for 5%-15% of peripheral blood lymphocytes, and are expressed in spleen, liver, iris, and lymph nodes. The phenotype of NK cells is CD3 - CD56 + , there are two isoforms of CD56 bright and CD56 dim ; CD56 bright The main function of NK cells is to secrete cytokines, CD56 dim NK cells are cytotoxic, accounting for 90% of peripheral blood NK cells. NK cells mainly induce and kill tumor cells and virus-infected ce...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2315C12N2501/515C12N2501/599
Inventor 葛淑娟徐矫健张道强刘传杰王玉娟刘鹏飞刘燕丽
Owner 青岛麦迪赛斯医疗技术有限公司
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