High-efficiency induction culture method for preparing natural killer NK cells

A NK cell and lethal technology, applied in the field of cells, can solve the problems of increasing risk and increasing the residual detection of purification steps, and achieve high safety, high quantity and high quality results

Pending Publication Date: 2020-09-22
暨赛国际再生医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the trophoblast cells used in the culture process are tumor cells, subsequent purification steps and residual detection need to be added, which also increases the risk of clinical use

Method used

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  • High-efficiency induction culture method for preparing natural killer NK cells
  • High-efficiency induction culture method for preparing natural killer NK cells
  • High-efficiency induction culture method for preparing natural killer NK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1. A method for efficiently inducing and culturing natural killer NK cells, comprising the following steps:

[0033] S1. Preparation stage: Coat cell culture flasks with recombinant human fibronectin and CD16 antibody: add 20 mL of D-PBS buffer to a 50 mL centrifuge tube, add recombinant human fibronectin: 8 μg / mL -15 μg / mL, CD16 antibody: 0.18ug / ml-0.22ug / ml; add the coating solution prepared above into a T175 cell culture flask, lay it flat, mix well, and coat overnight at 4°C in the dark;

[0034] S2. Peripheral blood mononuclear cell induction culture:

[0035] S21, collecting peripheral blood;

[0036] S22. Prepare autologous plasma and autologous serum;

[0037] S23. Separating the autologous plasma to obtain peripheral blood mononuclear cells, and preparing a cell suspension;

[0038] S24. Inoculating cells: add the cell suspension prepared in step S23 to the cell culture flask coated in step S1, add serum-free cell culture medium, add CD3 monoclona...

Embodiment 2

[0047] Example 2. A method for efficiently inducing and culturing natural killer NK cells, in addition to adding recombinant human fibronectin: 10 μg / mL-15 μg / mL in step S1, adding Her-2 and IFN-γ respectively in step S24 When the concentration reaches 0.15-0.22μg / ml, 0.5-1.0μg / ml, the final concentration of cytokines in the cell expansion stage is: IL-7 15-22 ng / mL, IL-21 3-8 ng / mL, etc. Except, all the other are identical with embodiment 1.

Embodiment 3

[0048] Example 3. A method for efficiently inducing and culturing natural killer NK cells, in addition to adding recombinant human fibronectin: 10 μg / mL-20 μg / mL in step S1, adding Her-2 and IFN-γ respectively in step S24 When the concentration reaches 0.12-0.20 μg / ml and 0.3-0.8 μg / ml, the final concentrations of cytokines added in steps S31 and S32 during cell expansion are: IL-2 10-12 ng / mL, IL-7 18-25 ng / mL, IL-15 46-50 ng / mL, IL-21 3-6 ng / mL and other differences, the rest are the same as in Example 1.

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Abstract

The present invention belongs to the technical field of cells and specifically relates to a high-efficiency induction culture method for preparing natural killer NK cells. The high-efficiency induction culture method for preparing natural killer NK cells mainly includes the following steps: S1, during a preparation stage, using a cell culture bottle coated with recombinant human fibronectin and CD16 antibody; S2, conducting peripheral blood mononuclear cell induction culture; S3, conducting expansion culture; and S4, using a trypan blue staining method and counting cells with a cell counting plate to calculate cell number and cell viability. The provided method can enable the NK cells to proliferate in large quantities, obtains the natural killer NK cells with high quantity, high viabilityand high quality, at the same time, has high safety and can meet needs of clinical treatment.

Description

technical field [0001] The invention belongs to the field of cell technology, and in particular relates to a method for efficiently inducing and culturing natural killer NK cells. Background technique [0002] Natural killer cells (NK) are important immune cells of the body. As the first line of defense of the body's defense system, they can not only exert their main tumor-killing effect in the natural immune system, but also secrete different Cytokines and various chemokines regulate the body's acquired immune response and are indispensable effector cells for the body to exert immune effects. Compared with αβT cells, γδT cells, CIK and other cells used for immunotherapy, NK cells have received more attention and attention due to their high cell killing activity, rapid onset of action, and not being restricted by MHC. Clinical application. At present, a large number of researchers at home and abroad have carried out research on the cultivation and biotherapy of human NK ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/078
CPCC12N5/0646C12N2500/90C12N2501/11C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/2318C12N2501/2321C12N2501/24C12N2501/515C12N2501/599C12N2506/11C12N2509/00C12N2533/52
Inventor 吴琨徐丽婉陈汉森肖桂琴罗瑞茵
Owner 暨赛国际再生医学科技有限公司
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