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Method for efficiently amplifying NK cells

A technology of NK cells and cells, applied in the field of cell culture, can solve the problem of weak killing ability of macrophages and B cells, and achieve the effect of enhancing killing function and promoting proliferation

Inactive Publication Date: 2017-05-31
SHANGHAI BIOMED UNION BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although K562 cells transfected with transmembrane IL-21 and CD137 complexes were used to activate and expand NK cells, although they had a strong ability to kill tumor cells, the killing effect on tumor cell-associated macrophages and B cells in the tumor microenvironment ability is weak

Method used

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  • Method for efficiently amplifying NK cells
  • Method for efficiently amplifying NK cells
  • Method for efficiently amplifying NK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] According to the method of the present invention, expand and cultivate NK cells with the following specific steps:

[0027] (1) Use the lentiviral transfection system to simultaneously transfect the genes of CD8α-interleukin 21, CD19, CD137L, CD86, and CD64 into K562 cells. CD8α is a membrane protein expressed on the cell membrane, and the CD8α gene is linked to the interleukin-21 gene so that Interleukin 21 is expressed on the cell membrane and becomes a transmembrane protein; CD19, CD137L, CD86, and CD64 are membrane proteins, and the vector contains a viral promoter and a selection marker gene; when the foreign expression vector enters the host cell, the promoter is activated, After being cultured for a period of time, transmembrane IL-21, CD19, CD137L, CD86, and CD64 can be expressed on the K562 cell membrane. The above cells were sorted by flow cytometry to obtain K562 cells stably expressing CD8αIL-21, CD19, CD137L, CD86, and CD64 as trophoblast cells of NK cells;...

Embodiment 2

[0036] The NK cells prepared in Example 1 were tested by flow cytometry, and the NK cells amplified by traditional IL-2 and K562 engineered cells were used as controls. Analyze the proportion of NK cells to the total cells (such as image 3 shown), the expression of Fc receptor CD16 on the surface of NK cells (such as Figure 4 shown), the expression of NK cell surface activation receptor NKG2D (such as Figure 5 shown) and the expression of the inhibitory receptor CD158α on the surface of NK cells (such as Figure 6 shown). image 3 , Figure 4 , Figure 5 and Figure 6 Show, utilize the NK cell that the method for the present invention prepares, the purity of NK cell (being CD3 - CD56 + ratio) was 97.8%, significantly higher than 86.1% of the control group; and the expression of CD16 on the surface of NK cells prepared by the method of the present invention was 88.7%, which was also significantly higher than 76.3% of the control group; in addition, using the method of...

Embodiment 3

[0038] The determination of NK cell killing rate was carried out according to the following operations:

[0039] (1) Target cell preparation: take K562 and MCF-7 cells in good growth state, centrifuge at 1000rpm for 5min, and count the cells; use RPMI-1640 complete medium to adjust the cell density to 2×10 5 / mL, spare;

[0040] (2) Preparation of effector cells: take NK cells in good growth state, centrifuge at 1500rpm for 5min, count the cells and adjust the cell density to 2×10 with RPMI-1640 complete medium 6 / mL, spare;

[0041] (3) Co-cultivation of target cells and effector cells: Add 10,000 target cells (ie 50uL) in a 96-well cell culture plate, and add effector cells (ie NK cells) at a ratio of 1:1, 5:1 and 10:1 , to a final volume of 100uL / well; and set blank control wells, target cell control wells and corresponding effector cell control wells, set 3 replicate wells for each well, and place the cell culture plate at 37°C, 5% CO 2 Cultivate in the incubator for 8 ...

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Abstract

The invention discloses a method for efficiently amplifying NK cells, in particular to a method for efficiently amplifying NK cells using K562 engineered cells of high expression membrane proteins CD19, CD137L, CD86, CD64 and transmembrane protein IL- 21 and combining with human IL-2 mutants. The method has the advantages of simple operation, low cost, large number of obtained NK cells, high purity and good killing effect; the method is suitable for large-scale preparation of the NK cells and lays a good foundation for the application of NK cell adoptive immunotherapy in clinical practice.

Description

technical field [0001] The invention relates to a cell culture method, in particular to a method for efficiently expanding NK cells. Background technique [0002] Natural killer cells (NK cells) are the third largest type of lymphocytes after T cells and B cells. They are the first line of defense of the human immune system and play an important role in the body's anti-viral infection and anti-tumor immunity. . NK cells recognize target cells without MHC restriction, and can kill tumor cells or virus-infected cells without prior sensitization. At the same time, it can also produce a series of cytokines to regulate the body's acquired immunity, which is a bridge connecting the body's innate immunity and specific immunity. NK cells have once become a new focus of immune cell adoptive therapy due to their direct killing activity and immunomodulatory activity. In addition, there are more and more reports that the primary NK cells or NK cell lines are genetically modified to i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0783C12N13/00
CPCC12N5/0646C12N5/0634C12N13/00C12N2501/2302C12N2502/30
Inventor 袁彩艳吴卫成刘根桃吴国祥柴勋
Owner SHANGHAI BIOMED UNION BIOTECHNOLOGY CO LTD
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