Amplification method of in-vitro NK cells and NK cells thereof

A technology of NK cells and nuclear cells, applied in the field of cell culture, can solve the problems of residual end products, different expansion potentials, and different NK cell contents, and achieve the effect of improving stability and strong killing ability.

Active Publication Date: 2021-12-07
河南省遗传资源细胞库有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the problem with this method is that the content of NK cells in blood samples from different sources is different, and the expansion potential is different, resulting in relatively large differences in amplification efficiency and amplification purity, which cannot meet the uniformity of batches in the development of cell drugs. sexual demands
The NK cells amplified by this method have the advantages of high amplification efficiency and high purity, but because the support used is a tumor cell line, although it has been irradiated and inactivated, it will inevitably appear in the final product. Residues in the drug will bring huge obstacles to the follow-up clinical research and drug registration application

Method used

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  • Amplification method of in-vitro NK cells and NK cells thereof
  • Amplification method of in-vitro NK cells and NK cells thereof
  • Amplification method of in-vitro NK cells and NK cells thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041]Embodiment 1: Separation and extraction of peripheral blood mononuclear cells The separation and extraction of peripheral blood mononuclear cells mainly includes the following steps:

[0042] (1) Take the peripheral blood and centrifuge at 2900r / min for 10min, take the upper plasma and inactivate it at 56°C for 30min, then centrifuge at 1500r / min for 10min, and take the supernatant as autologous plasma for later use.

[0043] (2) Use sodium chloride injection to dilute the remaining blood cell layer in step (1), and blow and mix well, take 30mL of diluted blood and slowly add it to the surface of 15mL lymphatic separation liquid, and centrifuge at 1500rpm / min for 20min; Aspirate the mononuclear cell layer into a new 50mL centrifuge tube, 15-20mL per tube, use sodium chloride injection to make up to 45mL, centrifuge at 1700rpm / min for 10min, remove the supernatant, and obtain crude mononuclear cells ;

[0044] (3) Second washing: resuspend the mononuclear cells obtained ...

Embodiment 2

[0047] A method for expanding NK cells in vitro, comprising the following steps:

[0048] (1) Preparation of magnetic microsphere suspension coupled with IL15 protein:

[0049] (S1) Mix His-tag protein purification magnetic beads with a diameter of 30 μm and made of polystyrene-nickel microspheres thoroughly and put them in a centrifuge tube for magnetic separation, discard the supernatant, and then add 10 mL of binding buffer (a mixture of 20mM sodium phosphate, 500mM sodium chloride and 5-50mM imidazole), resuspend the magnetic beads, then perform magnetic separation, remove the supernatant, and obtain pretreated His-tag protein purification magnetic beads;

[0050] (S2) Resuspend the His-tagged IL15 protein in 10 mL of binding buffer, then add it to the centrifuge tube containing the pretreated His-tag protein purification magnetic beads obtained in step (S1), and place the centrifuge tube in a vortex After oscillating for 20 seconds, place the centrifuge tube on a rotary ...

Embodiment 3

[0058] A method for expanding NK cells in vitro, comprising the following steps:

[0059] (1) Preparation of magnetic microsphere suspension coupled with IL15 and CD137L protein:

[0060] (S1) Mix His-tag protein purification magnetic beads with a diameter of 30 μm and made of polystyrene-nickel microspheres thoroughly and put them in a centrifuge tube for magnetic separation, discard the supernatant, and then add 10 mL of binding buffer (a mixture of 20mM sodium phosphate, 500mM sodium chloride and 5-50mM imidazole), resuspend the magnetic beads, then perform magnetic separation, remove the supernatant, and obtain pretreated His-tag protein purification magnetic beads;

[0061] (S2) Resuspend the His-tagged IL15 and CD137L proteins in 10 mL of binding buffer, and then add them to the centrifuge tube containing the pretreated His-tag protein purification magnetic beads obtained in step (S1). Place the centrifuge tube on a vortex shaker for 20 seconds, then place the centrifug...

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Abstract

The invention belongs to the technical field of cell culture, and particularly discloses an amplification method of in-vitro NK cells and the NK cells thereof. The amplification method of the in-vitro NK cells comprises the steps that mononuclear cells are separated and extracted from peripheral blood or umbilical cord blood, a culture medium containing protein coupled magnetic microspheres is adopted for carrying out amplification culture on the extracted mononuclear cells, and the NK cells are obtained, wherein at least one of IL15, IL18, CD86, CD137L and MICA is coupled to the coupled protein magnetic microspheres. According to the method, a plurality of activation and amplification signal protein molecules required by growth of the NK cells are coupled to the surfaces of the magnetic microspheres, and the protein coupled magnetic microspheres are used for in-vitro amplification culture of the NK cells, so that the amplification efficiency and purity stability of the NK cells in mononuclear cells of different sources can be remarkably improved; the NK cells obtained by amplification have relatively strong killing ability; and the exogenous magnetic bead microspheres can be completely removed from a culture system through a simple physical separation method, so that the control on the quality of a cell drug is facilitated.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to an in vitro NK cell expansion method and NK cells. Background technique [0002] NK cells are an important part of the innate immune system and an important immunoregulatory cell for the body to resist infection and prevent malignant transformation of cells. Different from the immune surveillance response system mediated by T cells, NK cells can directly kill tumor cells without tumor-specific antigen recognition, and are important effector cells for tumor immunotherapy. [0003] Obtaining sufficient quantity and purity of NK cells is the basis for developing NK cell drugs. At present, there are two main technical paths for large-scale expansion of NK cells: [0004] 1. Add a variety of cytokines such as IL2, IL15, IL18, etc. to the culture medium to stimulate NK cell activation signaling pathways and expand and activate NK cells. However, the problem with this method is ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2531/00C12N2533/50
Inventor 韦丹朱学义王迎耀薛晓峰秦志华
Owner 河南省遗传资源细胞库有限公司
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