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NK cell in-vitro proliferation method and culture medium

A technology for proliferation and NK cells in vitro, applied in the cultivation process, tissue culture, animal cells, etc.

Pending Publication Date: 2021-11-23
惠州腾大健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The effect of DFO on the proliferation and killing activity of NK cells has not been reported so far

Method used

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  • NK cell in-vitro proliferation method and culture medium
  • NK cell in-vitro proliferation method and culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] In vitro culture of peripheral blood NK cells

[0017] Extract 80mL of peripheral blood from healthy people, dilute with 100mL PBS buffer, use lymphocyte separation medium, centrifuge at 4°C for 15min, collect PBMC cells, wash with normal saline, and use complete medium (1640 medium + 10% FBS) to adjust PBMC cells The starting density is 2×10 6 / ml, the final concentration of IL-2 was 300U / ml, 37°C, 5% CO 2 cultivated under conditions.

[0018] Pay close attention to the cell density and growth status, replenish or change the fluid every other day to maintain the concentration of IL-2 at 300U / ml and the cell density at 2×10 6 / ml, cultivated for 10 days.

Embodiment 2

[0020] NK cell culture

[0021] Cultivate cells in a 96-well plate, get the NK cells cultivated in Example 1 for 10 days, set blank group (NK cell culture medium), control group (cell+NK cell culture medium) and DFO experimental group (cell+DFO-containing culture medium) Cell culture medium), 1 duplicate well in the blank group, 8 duplicate wells in the control group, and 32 duplicate wells in the DFO experimental group. 100 μL per well, 37°C, 5% CO 2 Conditioned for 24 hours. After 24 hours, the blank group and the control group were replaced with fresh NK cell medium to continue the culture, and the DFO group was replaced with the medium containing 10 μM, 40 μM, 80 μM, and 120 μM DFO to continue the culture for 48 hours.

[0022] Of the 8 replicate wells in the control group and the experimental group, 5 were used for proliferation activity experiments, and 3 were used for subsequent killing experiments.

Embodiment 3

[0024] NK cell proliferation activity assay

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Abstract

The invention relates to the field of cellular immunotherapy, in particular to an NK cell in-vitro proliferation method and a culture medium suitable for the method, and particularly relates to a cell culture medium taking deferoxamine as an effective component.

Description

technical field [0001] The invention relates to the field of cellular immunotherapy, in particular to a method for in vitro proliferation of NK cells and a culture medium suitable for the method, in particular to a cell culture medium with deferoxamine as an active ingredient. Background technique [0002] NK cells (natural killer cells) belong to non-specific immune cells and are important immune cells in the body. They can directly kill certain tumors and target cells infected by viruses without antigen pre-sensitization, so they are called natural killer cells. It can also secrete a variety of cytokines and chemokines to participate in immune regulation. Therefore, it plays an important role in the immune process of the body's anti-tumor and early anti-virus or intracellular parasitic infection. NK immune cell therapy has broad application prospects in antiviral and antitumor treatments. [0003] NK immune cell therapy is a kind of tumor immunotherapy in which NK immune...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/24
Inventor 黄咏晔
Owner 惠州腾大健康科技有限公司
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