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Method for efficient in-vitro amplification of peripheral blood NK cells

An in vitro expansion and NK cell technology, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of limited NK cell application, low NK cell content, low distribution frequency, etc., to reduce cellular immunity. The effect of originality, ensuring amplification multiple, and improving safety

Inactive Publication Date: 2018-09-04
CARBIOGENE THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, due to the extremely low content of NK cells in human peripheral blood and the low distribution frequency (<10%) in tumor tissues, they only account for 5%-10% of mononuclear cells, which greatly limits the use of NK cells as adoptive immune cells. clinical application
For many years, people have been trying to achieve large-scale expansion of NK cells in vitro. At present, after in vitro expansion and culture, NK cells can only be expanded several times or more than ten times, and the purity is not ideal, which cannot meet the practical application.

Method used

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  • Method for efficient in-vitro amplification of peripheral blood NK cells
  • Method for efficient in-vitro amplification of peripheral blood NK cells
  • Method for efficient in-vitro amplification of peripheral blood NK cells

Examples

Experimental program
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Effect test

Embodiment 1

[0036] A method for efficiently expanding peripheral blood NK cells in vitro, comprising the following steps:

[0037] 1) Under sterile conditions, take 10 mL of whole blood and anticoagulate it with heparin sodium to obtain anticoagulated whole blood, then dilute the anticoagulated whole blood with 10 mL of PBS, wherein the volume ratio of the whole blood to heparin sodium is 5000:10, wherein The concentration of heparin sodium is 6250U / mL.

[0038] 2) Take another 10 mL of lymphocyte separation medium, slowly spread the diluted anticoagulated whole blood on the surface of the lymphocyte separation medium, and keep the interface between the two liquid surfaces clear.

[0039] 3) Centrifuge the liquid obtained in step 2) at room temperature (use a horizontal rotor to centrifuge at a rate of 2000rpm for 15min), absorb a thin and dense buffy coat layer between the plasma layer and the separation liquid layer, and place the buffy coat layer on the In another centrifuge tube, blo...

Embodiment 2

[0043] A method for efficiently expanding peripheral blood NK cells in vitro, comprising the following steps:

[0044] 1) Under sterile conditions, take 10 mL of whole blood and anticoagulate it with heparin sodium to obtain anticoagulated whole blood, and then dilute the anticoagulated whole blood with 10 mL of PBS, wherein the volume ratio of the whole blood to heparin sodium is 5000:10, wherein The concentration of heparin sodium is 6250U / mL.

[0045] 2) Take another 10 mL of lymphocyte separation medium, slowly spread the diluted anticoagulated whole blood on the surface of the lymphocyte separation medium, and keep the interface between the two liquid surfaces clear.

[0046] 3) Centrifuge the liquid obtained in step 2) at room temperature (centrifuge at a speed of 2500 rpm for 13 min with a horizontal rotor), absorb a thin and dense buffy coat layer between the plasma layer and the separation liquid layer, and place the buffy coat layer on In another centrifuge tube, bl...

Embodiment 3

[0050] A method for efficiently expanding peripheral blood NK cells in vitro, comprising the following steps:

[0051] 1) Under sterile conditions, take 10 mL of whole blood and anticoagulate it with heparin sodium to obtain anticoagulated whole blood, and then dilute the anticoagulated whole blood with 10 mL of PBS, wherein the volume ratio of the whole blood to heparin sodium is 5000:10, wherein The concentration of heparin sodium is 6250U / mL.

[0052] 2) Take another 10 mL of lymphocyte separation medium, slowly spread the diluted anticoagulated whole blood on the surface of the lymphocyte separation medium, and keep the interface between the two liquid surfaces clear.

[0053] 3) Centrifuge the liquid obtained in step 2) at room temperature (use a horizontal rotor to centrifuge at a rate of 1500rpm for 17min), absorb a thin and dense buffy coat layer between the plasma layer and the separation liquid layer, and place the buffy coat layer on In another centrifuge tube, blo...

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Abstract

The invention relates to the technical field of cell amplification culture and discloses a method for efficient in-vitro amplification of peripheral blood NK cells. The method comprises the followingsteps: 1) performing anticoagulation on whole blood by heparin sodium, and diluting by PBS; 2) spreading the diluted anticoagulant whole blood on the liquid level of a lymphocyte separation liquid; 3)centrifuging, absorbing a white film layer, blowing, beating and uniformly mixing the white film layer, centrifuging, discarding the supernatant and repeatedly washing; 4) removing CD3+ lymphocytes,dyeing peripheral blood monocyte antibodies and sorting the cells; and 5) diluting the CD3+ lymphocytes, adding IL-2, IL-15, IL-21 and L-7 and performing amplification. The CD3+ cells are removed before amplification, so that the cellular immunogenicity is greatly reduced; meanwhile, four specific cell factors are used according to the specific proportion to activate and amplify the NK cells, so that the amplification multiple of the NK cells is guaranteed and the safety of the NK cells during treatment is greatly improved.

Description

technical field [0001] The invention relates to the technical field of cell expansion and culture, in particular to a method for efficiently expanding peripheral blood NK cells in vitro. Background technique [0002] NK cells, also known as natural killer cells, are large granular lymphocytes, derived from bone marrow, accounting for 5% to 15% of peripheral blood lymphocytes, and are important immune cells. Scientists first identified NK cells in 1975, which can directly kill tumor cells in the absence of T and B cells. Unlike cytotoxic CD8+ T cells, NK cells can directly kill MHC-negative tumor cells without pre-sensitization when killing tumor cells, which makes NK cells widely used in adoptive cell immunotherapy. [0003] However, due to the extremely low content of NK cells in human peripheral blood and the low distribution frequency (<10%) in tumor tissues, they only account for 5%-10% of mononuclear cells, which greatly limits the use of NK cells as adoptive immune...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/2321
Inventor 杨文君其他发明人请求不公开姓名
Owner CARBIOGENE THERAPEUTICS CO LTD
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