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Efficient in-vitro amplification method of human natural killer cells

A natural killer cell and high-efficiency technology, applied in the field of biomedicine, can solve the problems of high price, hidden safety hazards, low amplification efficiency, etc., achieve high production efficiency and ensure the effect of aseptic operation

Active Publication Date: 2016-04-06
THE SECOND HOSPITAL OF SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above methods of scholars at home and abroad have allowed NK cells to be expanded in large quantities, but the use of magnetic beads for separation has greatly increased the research cost, and with the help of K562, Wilms tumor cell line HFWT cells and other genetically modified tumor cells to stimulate NK expansion, Its security is at risk
Although the research methods of other scholars are easy to operate, the amplification efficiency is low.
At present, there are commercial NK cell isolation kits with an isolation purity of more than 95%, but they are expensive and difficult to meet the requirements of large-scale experiments and clinical tumor immunotherapy. Therefore, NK cell culture with simple operation and high expansion efficiency has been studied. technology is necessary

Method used

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  • Efficient in-vitro amplification method of human natural killer cells

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Embodiment 1

[0048] A method for high-efficiency expansion of human natural killer cells in vitro, comprising the following steps:

[0049] a. Prepare complete cell culture solution for use as a spare, to provide NK cells with the nutrients needed for their survival and expansion; the complete culture solution contains 2mM Gluta-MAX, 100μg / ml penicillin and streptomycin, and the rest is GMPCellGro R A mixture of DCSerum-freeMedium and fetal bovine serum; where GMPCellGro R The molar ratio of DCSerum-freeMedium to fetal bovine serum is 19:1;

[0050] b. Take 50ml of lymphocyte separation liquid at room temperature, pour 200ml of spare human peripheral blood onto the surface of the lymphocyte separation liquid, and centrifuge for the first time at 2500rpm for 15mins to form cell stratification;

[0051] c. Absorb the buffy coat cells in the cell layer obtained in step b into a new centrifuge tube, wash with phosphate buffer, and centrifuge for the second time at 2000 rpm for 5 mins to obtai...

Embodiment 2

[0059] A method for high-efficiency expansion of human natural killer cells in vitro, comprising the following steps:

[0060] a. Prepare complete cell culture solution for use as a spare, to provide NK cells with the nutrients needed for their survival and expansion; the complete culture solution contains 2mM Gluta-MAX, 100μg / ml penicillin and streptomycin, and the rest is GMPCellGro R A mixture of DCSerum-freeMedium and fetal bovine serum; where GMPCellGro R The molar ratio of DCSerum-freeMedium to fetal bovine serum is 4:1;

[0061] b. Take 300ml of lymphocyte separation liquid at room temperature, pour 200ml of spare human peripheral blood onto the surface of the lymphocyte separation liquid, and centrifuge for the first time at 1200rpm for 25mins to form cell stratification;

[0062] c. Absorb the buffy coat cells in the cell stratification obtained in step b into a new centrifuge tube, wash with phosphate buffer, and centrifuge for the second time at 800 rpm for 15 mins...

Embodiment 3

[0070] A method for high-efficiency expansion of human natural killer cells in vitro, comprising the following steps:

[0071] a. Prepare complete cell culture solution for use as a spare, to provide NK cells with the nutrients needed for their survival and expansion; the complete culture solution contains 2mM Gluta-MAX, 100μg / ml penicillin and streptomycin, and the rest is GMPCellGro R DCSerum-freeMedium and autologous serum mixture, wherein autologous serum accounts for 1% of the volume of the complete culture medium;

[0072] b. Take 100ml of lymphocyte separation liquid at room temperature, pour 200ml of spare human peripheral blood on the surface of the lymphocyte separation liquid, and centrifuge for the first time at 1800rpm for 20mins to form cell stratification;

[0073] c. Absorb the buffy coat cells in the cell stratification obtained in step b into a new centrifuge tube, wash with phosphate buffer, and centrifuge for the second time, the centrifugation condition is...

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Abstract

The invention discloses an efficient in-vitro amplification method of human natural killer (NK) cells. The efficient in-vitro amplification method includes the steps that CD16 monoclonal antibodies, ATG and IL-2 are added into a processed culturing system, incubation is carried out in an incubator at the temperature of 37 DEG C, counting and solution changing are carried out, and fresh IL-2 is added; a culture solution is changed at regular intervals, culturing continues for a proper time, and the human NK cells are obtained. The efficient in-vitro amplification method has the advantages that the NK cells can be efficiently amplified through the ATG and the CD16 antibodies in a short time without being subjected to separation of magnetic beads and irritation of tumor modification cells, production cost is greatly saved, and operation is easy and convenient; the number of amplification times of the NK cells is one thousand or more, and the final proportion ranges from 80% to 93%; production efficiency is high, and the efficient in-vitro amplification method can be entirely applied to large-scale clinical researching and the like; the growth process of the NK cells is completed under the GMP condition, sterile operation is guaranteed, various kinds of security detection are carried out at the final production stage, and the efficient in-vitro amplification method can be applied to clinical researching.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for high-efficiency expansion of human natural killer cells in vitro. Background technique [0002] Since the biological activity of NK cells was discovered in the 1880s, the research on NK cells and its application have been continuously carried out. Clinical trials at home and abroad have proved that NK cells are effective in treating hematological tumors, and it is expected to become one of the ways to completely cure hematological tumors. However, the clinical application of NK cells is also limited by their limited number. NK cells only account for 5%-15% of human peripheral blood lymphocytes. Therefore, the high-efficiency expansion of NK cells in vivo and in vitro is a matter of great concern to researchers and one of the bottlenecks in the widespread clinical application of NK cells. Only by solving the limitation of the number of NK cells, can the clinical...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 郑成云郭亚男
Owner THE SECOND HOSPITAL OF SHANDONG UNIV
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