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Genetically engineered cell and method for efficiently amplifying NK cells in vitro

A genetic engineering, NK cell technology, applied in the field of immunology, can solve the problems of uncertainty of NK cell culture, complex animal serum composition, introduction of mycoplasma, etc., to achieve the effect of low cost, large quantity, and enhanced lethality

Inactive Publication Date: 2018-11-16
武汉赛云博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above method can stimulate the efficient expansion of NK cells, the composition of animal serum is complex, and mycoplasma and viruses may be brought in during the sampling process, causing uncertainty and unsafety to NK cell culture

Method used

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  • Genetically engineered cell and method for efficiently amplifying NK cells in vitro
  • Genetically engineered cell and method for efficiently amplifying NK cells in vitro
  • Genetically engineered cell and method for efficiently amplifying NK cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of genetically engineered cells IL-15-IL-18-4-1BBL-K562 cells

[0041] (1) The IL-15, IL-18, and 4-1BBL genes were amplified by PCR, and then the respective amplification products were inserted into the XbaIBamHI site of the Piggybac Sleeping Beauty expression vector to construct PB-EF1-IL-15TM respectively -puro, PB-EF1-IL18TM-puro, PB-EF1-4-1BBL-puro transposons.

[0042] (2) Co-transfect K562 cells with PB-EF1-IL-15TM-puro transposon and transposase, and culture the transfected K562 cells; sort by flow cytometry to obtain IL-15 transmembrane Stably expressed K562 cells (IL-15-K562 cells).

[0043] (3) Co-transfect IL-15-K562 cells with PB-EF1-IL-18TM-puro transposon and transposase, and culture the transfected K562 cells; sort by flow cytometry to obtain IL -15. K562 cells stably expressing IL-18 across the membrane (IL-15-IL-18-K562 cells).

[0044] (4) Co-transfect IL-15-IL-18-K562 cells with PB-EF1-4-1BBL-puro transposon and transposase T...

Embodiment 2

[0045] Example 2: In vitro efficient expansion of NK cells

[0046] 1. Preparation of PBMC cells

[0047] Use an anticoagulant tube to collect peripheral blood from healthy people, and shake it while collecting to fully mix the peripheral blood with the anticoagulant; slowly add the anticoagulant blood to a 50ml centrifuge tube filled with an equal volume of lymphocyte separation medium (Ficoll), 450g, Centrifuge slowly for 25 minutes, and do not stop the centrifugation in the middle; after the centrifugation, carefully absorb the buffy coat cells above the lymphocyte separation solution, transfer to a new 50ml centrifuge tube, add PBS, 300g, and centrifuge slowly for 10 minutes , discard the supernatant, and keep the cell pellet at the bottom of the centrifuge tube; add PBS again, 160g, centrifuge slowly for 15min, discard the supernatant; finally add PBS, 300g, centrifuge slowly for 10min, discard the supernatant, and obtain PBMC cell.

[0048] 2. Expansion and culture of ...

Embodiment 3

[0057] Example 3: Determination of NK cell killing activity

[0058] The killing effect of NK cells on human leukemia cells K562 was detected by 4h LDH release assay.

[0059] 1. Take the passaged cell line K562 cells and count them to make 1×10 5 cells / mL cell suspension, add to 96-well plate, 50μl per well.

[0060] 2. Add the cultured NK cells into the 96-well plate according to the effect: target ratio of 1:1, 10:1, 20:1, and 40:1. At the same time, set the natural release holes for effector cells and target cells, and the natural release holes for the medium , target cell maximum release well, volume correction control, each well volume is 100μl, set 3 duplicate wells, centrifuge at 250g for 4min, place at 37°C, 5% CO 2 , 95% saturated humidity incubator for 4 hours.

[0061] 3. 45 minutes before the end of the reaction, add 10 μl of lysate to each well of the target cell maximum release well. After the reaction, pipette 50 μl supernatant and 50 μl LDH enzyme reaction...

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Abstract

The invention relates to specifically relates to a genetically engineered cell and a method for efficiently amplifying NK cells in vitro by using the genetically engineered cell, belonging to the field of immunology. According to the invention, a Piggybac transposon system is utilized to construct a K562 engineered cell that realizes transmembrane stable expression of IL-15, IL-18 and 4-1BBL; andthe method for amplifying and activating NK cells based on the genetically engineered K562 cell is provided in virtue of an in-vitro cell stimulation technology. The genetically engineered cell can realize simultaneous transmembrane high-expression of IL-15, IL-18 and 4-1BBL. The NK cell amplification method of the invention is simple to operate and low in cost; the amplified NK cells have the advantages of high purity, large quantity, good killing activity, etc.; and the NK cell amplification method is suitable for large-scale production of NK cells and lays a good foundation for the clinicalapplication of adoptive immunotherapy of NK cells.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a genetically engineered cell and a method for efficiently expanding NK cells in vitro using the genetically engineered cell. Background technique [0002] The latest edition of the "World Cancer Report" comprehensively describes and analyzes the overall situation and prevalence trends of 28 cancers in more than 180 countries around the world. The report predicts that global cancer cases will show a rapid growth trend. 14 million cancer cases and 8.2 million deaths. Among them, China added 3.07 million cancer patients and caused about 2.2 million deaths, accounting for 21.9% and 26.8% of the global total respectively. Among them, lung cancer, gastric cancer, and liver cancer are the cancers with the highest incidence rates, while breast cancer, colorectal cancer, and cervical cancer threaten women's health. [0003] Tumor immunotherapy is recognized as the fourth major tumor treatment...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/0783
CPCC07K14/54C07K14/5443C07K14/705C12N5/0646C12N2501/999C12N2502/30
Inventor 许先进韩俊峰雷菁曾祺森刘慧莹
Owner 武汉赛云博生物科技有限公司
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