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A method for amplifying cord blood nk in vitro

An in vitro amplification and umbilical cord blood technology, applied in biochemical equipment and methods, blood/immune system cells, microorganisms, etc., can solve the problems of umbilical cord blood NK amplification method complex, long in vitro amplification time, and low cell purity , to avoid the risk of tumor induction, shorten the time of in vitro amplification, and achieve good killing effect

Active Publication Date: 2021-05-28
天晴干细胞股份有限公司
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AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problems that the existing umbilical cord blood NK amplification method is complicated, high in cost, low in safety due to the introduction of animal source components, long in vitro amplification time, and low in cell purity, and provides an in vitro amplification Cord blood NK method

Method used

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  • A method for amplifying cord blood nk in vitro
  • A method for amplifying cord blood nk in vitro
  • A method for amplifying cord blood nk in vitro

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specific Embodiment approach 1

[0035] Specific embodiment one: a kind of method of expanding umbilical cord blood NK in vitro of the present embodiment, its specific steps are as follows:

[0036] 1. Add coating solution and normal saline in a volume ratio of 1:4 in a T75 culture flask; 1 mL of coating solution contains components: CD16 monoclonal antibody at a concentration of 3-7 μg / mL, and a concentration of 800-1200 μg / mL mL of IFN-γ and GM-CSF with a concentration of 400-600 IU / mL; shake the cell bottle to make the coating solution cover the bottom of the bottle, let stand at room temperature for 1 hour, remove the coating solution, wash the bottom of the bottle with normal saline once, remove Physiological saline, placed in a T75 culture bottle for later use;

[0037] 2. Activation of mononuclear cells in cord blood

[0038] A. Isolation of mononuclear cells:

[0039]Divide 80-100mL of umbilical cord blood into centrifuge tubes, centrifuge at 650g for 15 minutes at room temperature, remove the upper...

specific Embodiment approach 2

[0048] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is that 1 mL of coating solution contains components: CD16 monoclonal antibody with a concentration of 5 μg / mL, IFN-γ with a concentration of 1000 μg / mL and 500 IU / mL GM-CSF. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0049] Embodiment 3: This embodiment differs from Embodiment 1 in that the volume ratio of the lymphocyte separation solution to the diluted cord blood is 15:50. Others are the same as in the first embodiment.

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Abstract

A method for amplifying cord blood NK in vitro, which relates to a method for amplifying cord blood NK. The purpose of the present invention is to solve the problem that the existing cord blood NK amplification method is complicated and costly. Due to the introduction of animal source components, it is safe Due to the problems of low stability, long in vitro expansion time and low cell purity, the present invention promotes the proliferation of umbilical cord blood NK cells and enhances the activity of NK cells by adding a variety of cytokines. The invention is simple in operation, low in cost, does not introduce animal source components, and has high safety. The umbilical cord blood NK cells can be harvested after 15 days of culture in the present invention, and the cell purity is 95.33%, the total number of cells is 12 billion / part of umbilical cord blood, and the killing effect on lung cancer A549 cells is good.

Description

technical field [0001] The invention relates to a method for expanding umbilical cord blood NK in vitro. Background technique [0002] For nearly a decade, surgery and chemotherapy have been used as the main methods of eliminating tumors. However, the body's resistance to anticancer drugs leads to a significantly higher chance of tumor recurrence. Therefore, there is an urgent need to study effective methods to solve the problem of tumor recurrence. Recent studies have shown that the immune system plays an important role in suppressing the onset of malignant tumors. Natural killer cells (Nature killer cells, NK) are derived from hematopoietic precursor cells in the bone marrow and are widely distributed in different tissues, such as peripheral blood, spleen, lung, liver and uterus. NK cells account for 5%-10% of lymphocytes in the peripheral blood of normal people, and 10%-20% of lymphocytes in cord blood. A large number of studies have confirmed the role and therapeutic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/90C12N2501/22C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/2321C12N2501/24C12N2501/599
Inventor 李妍张怡刘春香翟莹刘艳青
Owner 天晴干细胞股份有限公司
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