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Method for in-vitro amplification of NK cells and NK cells obtained by same

A technology of NK cells and in vitro expansion, applied in the field of NK cells, can solve the problems of high preparation cost and difficult expansion of NK cells in vitro, and achieve the effect of reducing the amount of use

Inactive Publication Date: 2016-07-13
TIANJIN PURUI SAIER BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the biggest problem that plagues the large-scale application of NK cells is the difficulty of their in vitro expansion and the high cost of preparation.
Although the method of using tumor trophoblasts to promote the expansion of NK cells can obtain a large number of high-purity cells, the safety problem is the threshold that cannot be bypassed.

Method used

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  • Method for in-vitro amplification of NK cells and NK cells obtained by same
  • Method for in-vitro amplification of NK cells and NK cells obtained by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] 1) Preparation of NK cell complete medium: immune cells containing FBS with a volume fraction of 6%, and IL-2 with a final concentration of 1000IU / ml, IL-12 with a final concentration of 50IU / ml, and IL-15 with a final concentration of 50IU / ml Culture medium;

[0058] 2) Prepare CD16Mab coating solution with NK cell complete medium: NK cell complete medium contains 8ug / ml CD16Mab;

[0059] 3) Add 5ml of CD16Mab coating solution to the T-175 cell culture flask, and coat for 24 hours;

[0060] 4) The PBMCs obtained from peripheral blood were separated according to 5*10 5 Inoculate the cells at a final concentration of / ml into the pre-coated cell culture flask, add NK cell complete medium until the culture system is 50ml, add IFN-r according to the final concentration of 1000IU / ml, and start the induction culture;

[0061] 5) On the fourth day of induction culture, add 50ml NK cell complete medium to the culture system to continue the culture;

[0062] 6) On the sixth ...

Embodiment 2

[0072] 1) Preparation of NK cell complete medium: immune cells containing FBS with a volume fraction of 5%, and IL-2 with a final concentration of 500IU / ml, IL-12 with a final concentration of 20IU / ml, and IL-15 with a final concentration of 20IU / ml Culture medium;

[0073] 2) Prepare CD16Mab coating solution with NK cell complete medium: NK cell complete medium contains 4ug / ml CD16Mab;

[0074] 3) Add 1ml of CD16Mab coating solution to the T-75 cell culture flask, and coat for 4 hours;

[0075] 4) The PBMCs obtained from peripheral blood were separated according to 3*10 5 Inoculate cells at a final concentration of 500IU / ml into cell culture flasks coated in advance, add NK cell complete medium to a culture system of 20ml, add IFN-r at a final concentration of 500IU / ml, and start induction culture;

[0076] 5) On the fourth day of induction culture, add 20ml NK cell complete medium to the culture system to continue the culture;

[0077] 6) On the seventh day of culture, ta...

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Abstract

The invention discloses a method for in-vitro amplification of NK cells and the NK cells obtained by the same. The method comprises the following steps: taking peripheral blood mononuclear cells (PBMC) separated out from peripheral blood as seed cells, adding the cells into a culture bottle pre-coated with CD16Mab when induced culture begins and enlarged culture is carried out every time, performing continuous stimulation on the cells, and finally obtaining a large amount of high-purity NK cells. Under the situation that the stimulation is performed without using trophoblast cells, the large amount of high-purity NK cells is finally obtained through the method of performing continuous stimulation by coating the culture bottle with CD16Mab, and the use amount of cell factors is reduced.

Description

technical field [0001] The present invention relates to a method for expanding NK cells in vitro, especially using peripheral blood mononuclear cells (PBMC) as seed cells, by adding the cells to the pre-coated CD16Mab culture at the beginning of induction culture and each expansion culture In the flask, the method for continuously stimulating cells and finally preparing a large amount of high-purity NK cells and the obtained NK cells. Background technique [0002] Tumor is currently the most serious disease plaguing human beings, ranking first in both morbidity and death. As the fourth means of tumor treatment, tumor immunotherapy is gradually being accepted by people. As a very important part of tumor immunotherapy technology, NK cells have the advantages of strong cytotoxicity, rapid onset of effect and no antigen inhibition. received widespread attention. Moreover, in addition to the function of killing tumor cells, NK cells also have the unique property of anti-viral i...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2302C12N2501/2312C12N2501/2315C12N2501/24C12N2501/599
Inventor 鲁振宇韩洪起刘俊江张冰晶秦臻徐悦黄文敬
Owner TIANJIN PURUI SAIER BIOLOGICAL TECH CO LTD
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