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In-vitro expansion culture method of NK cells

A technology of NK cells and in vitro expansion, applied in the fields of bioengineering and biomedicine, can solve the problems of poor killing activity and low NK cell expansion rate, and achieve the effect of simplifying steps and improving killing activity

Inactive Publication Date: 2017-05-24
SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is to provide an in vitro expansion method for NK cells that can improve the expansion rate and killing activity of NK cells in view of the defects of low NK cell expansion rate and poor killing activity in the expansion of NK cells in the prior art. increase method

Method used

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  • In-vitro expansion culture method of NK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The in vitro expansion and culture method of NK cells provided by the invention comprises the following steps:

[0037] S1a, obtaining NK cells;

[0038] S11a, isolation of mononuclear cells;

[0039] Collect 100ml of human peripheral blood or 80mL of neonatal umbilical cord blood, dilute the blood sample with 1-2 times the volume of PBS solution, mix thoroughly to obtain a PBS blood sample mixture, and then add it to each 50ml centrifuge tube 15ml of Ficoll separation solution, the density of Ficoll separation solution is 1.077g / mL; then slowly add 20-30ml of PBS blood sample mixture, centrifuge at room temperature, adjust the speed of the centrifuge to 0, and centrifuge at 20°C and 400g for 30min. The middle layer is mononuclear cells; use a Pasteur pipette to carefully absorb, then fill up the centrifuge tube with 1×PBS, wash the mononuclear cells by sufficient centrifugation (400g, centrifuge for 10min), resuspend the cells, and set aside.

[0040] S12a, CD34 + S...

Embodiment 2

[0045] The difference from Example 1 is that the concentrations of the added hematopoietic stem cell growth factor and IGF-1 in the stem cell culture medium in this example are respectively: the concentration of stem cell factor is 10 ng / ml, and the concentration of FLT-3L is 20 ng / ml and the concentration of interleukin-15 was 20 ng / mL and the concentration of IGF-1 was 50 ng / mL.

Embodiment 3

[0047] The difference from Example 1 is that the concentrations of the added hematopoietic stem cell growth factor and IGF-1 in the stem cell culture medium in this example are respectively: the concentration of stem cell factor is 50 ng / ml, and the concentration of FLT-3L is 80 ng / ml and the concentration of interleukin-15 was 80 ng / mL and the concentration of IGF-1 was 150 ng / mL.

[0048] In order to further verify the remarkable effect of the NK cell in vitro expansion and culture method provided by the present invention, the following experiments were carried out for detection and verification.

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Abstract

The invention relates to an in-vitro expansion culture method of NK cells. The culture method comprises the following steps that the NK cells are acquired; the NK cells are subjected to in-vitro expansion culture in a culture medium containing an insulin-like growth factor-1. The insulin-like growth factor-1 replaces feeder cells for carrying out expansion culture on the NK cells, so that the steps of NK cell expansion culture are simplified; the cell viability, the multiplication rate and the killing activity of the NK cells can be improved through the insulin-like growth factor-1, the insulin-like growth factor-1 can stimulate the NK cells to secrete the endogenic insulin-like growth factor-1 to further improve the killing activity of the NK cells, and therefore the defects that in the prior art, due to the fact that the feeder cells are used for carrying out multiplication culture on the NK cells, the NK cell multiplication rate is low, and the killing activity is poor are overcome.

Description

technical field [0001] The invention relates to the fields of bioengineering and biomedicine, in particular to a method for expanding and culturing NK cells in vitro. Background technique [0002] Natural killer cells (NK cells), as a large group of lymphocytes juxtaposed with T cells and B cells, are important members of the innate immune system. In addition to participating in the innate immune response, NK cells also play an important regulatory role in the immune regulation of adaptive immunity. The target cells of NK cells mainly include certain tumor cells (including some cell lines), virus-infected cells, some self-tissue cells (such as blood cells), parasites, etc. Therefore, NK cells are an important immune system for the body to fight tumors and infections. Factors are also involved in type II hypersensitivity reactions and graft-versus-host reactions. In summary, the ex vivo expansion and activation of NK cells is the key to promoting frequent clinical applicati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 曾宪卓鲁菲
Owner SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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