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Efficient factor secreting type NK cell amplification method and application thereof

A NK cell and secretory technology, applied in cell dissociation methods, animal cells, vertebrate cells, etc., can solve the problems of low number and purity of NK cells, achieve efficient survival and expansion, increase the number of expansion, guarantee The effect of uniformity

Active Publication Date: 2021-09-21
天晴干细胞股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention is to solve existing methods to amplify CD56 bright The number and purity of NK cells are low, providing an efficient factor-secreting NK cell expansion method and its application

Method used

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  • Efficient factor secreting type NK cell amplification method and application thereof
  • Efficient factor secreting type NK cell amplification method and application thereof
  • Efficient factor secreting type NK cell amplification method and application thereof

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Experimental program
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Effect test

specific Embodiment approach 1

[0027] Specific embodiment one: the efficient method for expanding factor-secreting NK cells in this embodiment includes the following steps:

[0028] Step 1: Separating mononuclear cells from umbilical cord blood or peripheral blood;

[0029] Step 2: Sorting mononuclear cells with a sorting flow cytometer, the screening range is the uppermost 5% of the Q1 area, that is, CD56 strongly positive cells are obtained;

[0030] Step 3: Culture the strongly positive CD56 cells obtained in Step 2 at 37°C and 5% CO 2 Saturated humidity; the specific method is: inoculate CD56 strongly positive cells into a T25 bottle containing serum-free medium A for culture, collect the cells on the 3rd day of culture, and supplement them with serum-free medium A to continue the culture; The cells were transferred to T75 culture flasks at 5 days, and serum-free medium A was supplemented; after that, the liquid was replenished every 2 days, and the replenished liquid was GT-T551 H3 serum-free medium c...

specific Embodiment approach 2

[0038] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the method for separating mononuclear cells in cord blood or peripheral blood in step 1 is: take a portion of cord blood or peripheral blood, centrifuge in a centrifuge tube, remove After the upper layer of light yellow plasma, use normal saline to replenish the original volume of the plasma, then take a new centrifuge tube, add lymphocyte separation medium, and add the diluted blood to the lymphocyte separation medium, and separate the diluted blood from the lymphocytes The liquid is layered and centrifuged. The lower layer is red blood cells, and the middle buffy coat is mononuclear cells. After removing part of the supernatant, suck the buffy coat into a new centrifuge tube, add an equal volume of normal saline, and centrifuge to remove the supernatant ,washing. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0039] Embodiment 3: This embodiment differs from Embodiment 1 or Embodiment 2 in that: the Q1 region described in Step 2 is NK cells. Others are the same as in the first embodiment.

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Abstract

The invention discloses an efficient factor secreting type NK cell amplification method and application thereof, and relates to a factor secreting type NK cell amplification method and application thereof, in particular to a CD56light NK cell amplification method and application thereof, and aims at solving the problems that the number and the purity of the CD56light NK cells amplified by an existing method are low. The method comprises the following steps: 1, separating mononuclear cells in cord blood or peripheral blood; 2, sorting the mononuclear cells by using a sorting type flow cytometer; 3, culturing the sorted cells, inoculating CD56 strongly positive cells into a T25 bottle for culturing, collecting the cells when the cells are cultured for the third day, and supplementing the cells into a serum-free culture medium for continuous culture; when the culture is carried out for the 5th day, transferring into a T75 culture bottle, and supplementing a serum-free culture medium; and supplementing liquid every two days, and culturing to the 15th day. The factor secreting type NK cell prepared by the method can be used for resisting inflammation. The method is applied to the field of factor secreting type NK cell amplification.

Description

technical field [0001] The present invention relates to a factor-secreting NK cell expansion method and its application, in particular to CD56 bright NK cell expansion method and its application. Background technique [0002] Natural killer (NK) cells are an important class of lymphocytes and the first natural defense line of human innate immunity. Due to its important role in xenograft immunity, control of tumor growth and virus infection, it has received more and more attention in the fields of biotechnology and cell medicine research in recent years. According to the expression level of CD56 molecule, NK cells can be divided into CD56 bright NK, CD56 dim NK cells and CD56 neg 3 subpopulations, in the peripheral blood of healthy people, CD56 dim NK cells account for 90% of NK cells in peripheral blood, mainly with killing function, CD56 bright NK cells account for 10% of peripheral blood NK cells, mainly secrete cytokines, and there is no or only a very small amo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P29/00
CPCC12N5/0646A61K35/17A61P29/00C12N2509/00C12N2500/90C12N2501/2302C12N2501/2315C12N2501/2321C12N2500/34Y02A50/30
Inventor 刘春香张怡吕秀明刘艳青
Owner 天晴干细胞股份有限公司
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