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In-vitro three-dimensional amplification culture method for NK cells

A technology of NK cell expansion and culture, applied in the fields of bioengineering and biomedicine, can solve the problems of low NK cell expansion rate and poor killing activity, and achieve the effect of improving cell activity, promoting contact, and solving poor NK cell activity

Inactive Publication Date: 2016-03-30
SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The technical problem to be solved by the present invention is to provide a method 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 two-dimensional environment in the prior art. In vitro three-dimensional expansion and culture method of NK cells

Method used

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  • In-vitro three-dimensional amplification culture method for NK cells
  • In-vitro three-dimensional amplification culture method for NK cells

Examples

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Effect test

Embodiment 1

[0051] The in vitro three-dimensional expansion culture method of NK cells provided by the invention comprises the following steps:

[0052] S1a, obtaining NK cells;

[0053] S11a, isolation of mononuclear cells;

[0054] 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.

[0055] S...

Embodiment 2

[0062] The difference from Example 1 is that the concentrations of the added hematopoietic stem cell growth factors in the stem cell culture medium in this example are: the concentration of interleukin-2 is 100 U / ml, the concentration of interleukin-12 is 0.5ng / ml ml and the concentration of interleukin-15 was 2 ng / mL and that of interleukin-18 was 5 ng / mL.

Embodiment 3

[0064] The difference from Example 1 is that the concentrations of the added hematopoietic stem cell growth factors in the stem cell medium in this example are: the concentration of interleukin-2 is 300 U / ml, the concentration of interleukin-12 is 4.5ng / ml ml and the concentration of interleukin-15 was 8 ng / mL and that of interleukin-18 was 15 ng / mL.

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Abstract

The invention relates to an in-vitro three-dimensional amplification culture method for NK cells. The culture method comprises the following steps: obtaining NK cells; inoculating a three-dimensional culture bracket with the NK cells; and performing in-vitro three-dimensional amplification culture, wherein the three-dimensional culture bracket is a chitosan-sodium alginate bracket. Through the porous structure as well as the mechanical properties and biocompatibility of the chitosan-sodium alginate bracket, the growing space of the NK cells and the contact area with nutrient substances are enlarged, and the NK cells can be uniformly distributed in the chitosan-sodium alginate bracket, thus the cell activity of the NK cells can be improved, and the improvement of the amplification rate and killing activity of the NK cells is facilitated; and the defects such as low activity and low amplification rate of NK cells caused by culturing the NK cells with feeder cells in a two-dimensional environment in the prior art are solved.

Description

technical field [0001] The invention relates to the fields of bioengineering and biomedicine, in particular to an in vitro three-dimensional expansion culture method of NK cells. Background technique [0002] Natural killer cells (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 pro...

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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