NK cell in-vitro amplification culture method

A technology of NK cell and in vitro expansion, which is applied in the field of NK cell in vitro expansion and culture, and can solve the problems of unfavorable large-scale NK cell culture and clinical research, low number of expansion numbers and purity, and increased cost of NK cells. It achieves the effect of being conducive to large-scale production, strong amplification ability and simple method

Inactive Publication Date: 2020-07-10
GUANGDONG XIANKANGDA BIOTECH CO LTD
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The safety of clinical application of the first two methods of expanding NK cells has not been verified, and there is a certain risk in using K562 cells as tumor cells, so these two methods are mostly used in scientific research
The third method using factors to stimulate the expansion and culture of NK cells has better safety and high clinical application value, but the expansion number and purity are not high and unstable
Now, researchers at home and abroad have discovered that removing T cells from mononuclear cells is more conducive to the expansion of NK cells. This method can directly make NK cells into a dominant cell group and stimulate and induce the expansion of NK cells faster. This method uses magnetic beads, which greatly increases the cost of NK cell expansion, which is not conducive to large-scale NK cell culture and clinical research

Method used

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  • NK cell in-vitro amplification culture method

Examples

Experimental program
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Effect test

Embodiment 1

[0021] The method of NK in vitro expansion and culture in this example uses the peripheral blood of volunteers to carry out the expansion experiment, and goes through culture bottle coating, blood collection, isolation of mononuclear cells and plasma inactivation, primary screening to remove T cells, stimulation induction, and expansion. Culture, harvest cells and assay. The specific steps of the method for the NK in vitro expansion and cultivation are as follows:

[0022] (1) Coating treatment of culture bottle: use 10ml of coating solution containing CD16 concentration of 10μg / ml to cover the TC-treated culture bottle, place it in a refrigerator at 4°C, and incubate overnight; after the coating is completed, discard the coating solution and use 10ml Washed twice with PBS, set aside;

[0023] (2) Collect peripheral blood from volunteers and obtain PBMC mononuclear cells by gradient centrifugation: the tests for hepatitis B surface antigen, hepatitis C antibody, human immunod...

Embodiment ２

[0029] The method for in vitro expansion and culture of NK in this example is to collect cord blood from the hospital (informed by the puerpera), which is consistent with the method for in vitro expansion and culture of NK in peripheral blood. Since the content of NK in cord blood is lower, the expansion and culture The amount of stimulation and expansion culture factors in the medium was different. The specific steps of the method for the NK in vitro expansion and cultivation are as follows:

[0030] (1) Coating treatment of culture flasks: Cover TC-treated culture flasks with 10ml of coating solution containing CD16 concentration of 20μg / ml, place in 4°C refrigerator, and incubate overnight; after coating, discard the coating solution and use Wash twice with 10ml PBS, set aside;

[0031] (2) Gradient centrifugation of umbilical cord blood from the hospital to obtain mononuclear cell PBMC: the detection of hepatitis B surface antigen, hepatitis C antibody, human immunodefici...

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Abstract

The invention relates to an NK cell in-vitro amplification culture method, and belongs to the field of cell culture. The NK cell in-vitro amplification culture method comprises the following steps: screening out a large number of T cells by using a CD 16-coated culture bottle, so that NK cells become dominant cell populations; stimulating and inducing the NK cells by using IL-2, IL-12, IL-15, IL-18, SCM and IFN-gamma; and amplifying by using a serum-free complete medium containing IL-2 to obtain a large number of high-purity NK cells. The problem that a large number of high-purity NK cells cannot be obtained by amplification in the prior art is solved. According to the method, most of T cells are screened out by using the CD 16-coated culture bottle, so that the NK cells become dominant cells, and the NK cells are stimulated, induced and amplified; the method is simple to operate, easy to implement and high in amplification efficiency, no feeder cells or conductor serum is added in theculture process, the medical ethics and GVHD risk problems cannot be caused, and the method is suitable for clinical experimental research and large-scale preparation of the NK cells.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for expanding and culturing NK cells in vitro. Background technique [0002] Tumor is a major disease that plagues human health, but due to its high complexity, diversity, variability and heterogeneity, a more in-depth treatment method has not been found. With the advancement of medicine, the cure rate and survival rate of cancer Rates have improved significantly, but it is still a refractory disease. At present, the treatment methods for cancer at home and abroad include surgical resection, chemotherapy and radiation therapy. Scientists have invented adoptive immunotherapy for cancer patients who cannot autonomously produce more effective killing effects. Adoptive immunotherapy is a hotspot in the research of tumor treatment, and it has been widely used in clinic. [0003] NK cells, also known as natural killer cells, are the third type of lymphocytes except T a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/599
Inventor 谢海涛方晓薛卫巍刘元甲刘家飞翟秋梅陈坤
Owner GUANGDONG XIANKANGDA BIOTECH CO LTD
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