Improved method for cultivating NK (natural killer) cells

A technology of NK cells and culture methods, applied in the field of improved NK cell culture, can solve the problems of insufficient purity, lack of activation effect, and inability to achieve the amount required for therapeutic reinfusion, and achieve fast expansion and purity. high effect

Inactive Publication Date: 2016-08-31
王晓冰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even if NK cells are shown in sufficient numbers, without activating them, their work efficiency is very low
[0003] At present, a large number of researchers at home and abroad have carried out research on the cultivation and biological treatment of human NK cells, but most of the researches are purely using interleukin cytokines such as IL-2 and IL-12 to cultivate, but the results obtained in this way As a result, there are often two disadvantages, either the quantity cannot be increased, and the amount required for therapeutic reinfusion cannot be achieved, or the purity cannot be increased, and the desired effect cannot be achieved.
Because none of the interleukin cytokines are strong NK cell activators, they lack a strong activation effect

Method used

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  • Improved method for cultivating NK (natural killer) cells
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  • Improved method for cultivating NK (natural killer) cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Comparison of the cell amplification factor between this method and the conventional method

[0028] In the present embodiment, the experimental group isolates and cultivates the cell culture method of NK cells, and the specific steps are as follows:

[0029] Step 1. Take 60ml of heparin-anticoagulated venous blood and divide it into 2 branches to get mononuclear cells (PBMC). The specific steps are as follows: add 15ml of lymphocyte separation solution to the 50ml centrifuge tube, and then stick to the tube wall, slowly add 30ml of venous blood to avoid damaging the water phase of the lymphocyte separation solution. 400g, centrifuge for 20 minutes. Carefully aspirate the upper layer of plasma into a new 50ml centrifuge tube, heat at 56°C for 25 minutes to fully inactivate complement, and set aside. Aspirate the buffy coat layer in the middle layer, that is, the mononuclear cell layer, into a new 15ml centrifuge tube, wash thoroughly with 0.01M PBS 3 times, centrif...

Embodiment 2

[0036] 1. Comparison of cell purity between this method and conventional methods

[0037] The experimental subjects of this embodiment were divided into two groups, the experimental group and the conventional group. Among them, the experimental group cultured NK cells according to the cell culture method in Example 1. The conventional group was cultivated according to traditional techniques. On the 18th day, the cell culture fluid was taken from the two groups of culture systems, washed twice with PBS, and the cell concentration was adjusted to 2X10 after washing. 5 / ml, add flow cytometry antibody, 4C in the dark for 30min, wash off excess antibody, resuspend in PBS, and perform flow cytometry detection of cell phenotype. The antibodies used were from BD Company in the United States, and the results are shown in Table 2. The CD3 of the conventional group - CD56 + NK cells accounted for 50.16±1.02%, CD3 in this method - CD56 + NK cells accounted for 82.86±1.83%, and ther...

Embodiment 3

[0042] 1. The secretion level of IL2, IL12, IFN-γ secreted by cells secreted by this method and the conventional method is compared

[0043] The experimental subjects of this embodiment were divided into two groups, the experimental group and the conventional group. Among them, the experimental group cultured NK cells according to the cell culture method in Example 1. The conventional group was cultivated according to traditional techniques. On the 18th day, 100 ml of cell culture fluid was taken from the two groups of culture systems, concentrated by conventional methods, and the contents of IL2, IL12, and IFN-γ were detected with kits. All kits were purchased from Wuhan Boster Bioengineering Co., Ltd. . The experimental results are shown in Table 3. The levels of IL2, IL12 and IFN-γ secreted by cells in the experimental group were significantly higher than those in the conventional group (unit μg / ml).

[0044] group

IL2

IL12

IFN-γ

regular group...

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Abstract

The invention discloses an improved method for cultivating NK (natural killer) cells. The improved method has the advantages that sufficient quantities of the NK cells can be cultivated by the aid of the improved method in a separation manner, the improved method is high in amplification speed, and 70 billion cells can be generally cultivated in peripheral blood in 15 days; acetylated alpha glucan which is used as an activating mobilizing agent is added into media, accordingly, the NK cells can be successfully activated and can be promoted to secrete IL2, IL12 and IFN-gamma, critical effects for maintaining the activity of the NK cells and the amplification rates after the NK cells are activated can be realized by the IL2, the IL12 and the IFN-gamma, and the improved method has clinical application value; the NK cells obtained by the aid of the improved method are high in purity, as discovered via analysis on detection results of flow cell meters, the CD3<->CD56<+> NK cell population can reach 80% at least, and accordingly clinical application requirements can be met.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to an improved NK cell culture method. Background technique [0002] Natural killer cells (natural killer cells, NK) are important immune cells in the body, mainly distributed in peripheral blood, accounting for 5%-10% of PBMC, lymph nodes and bone marrow also have NK activity, but the level is lower than peripheral blood. NK cells are a group of heterogeneous and multifunctional immune cells, one of the many immune cells in humans, and they are killer cells with natural cytotoxic activity. The main function is to eliminate viruses, bacteria, cancer cells, etc. that attack the human body. The way it works is divided into: direct killing, releasing perforin, NK cytotoxic factor and TNF, etc. There are at least 5 billion NK cells in each person's body, and as many as 100 billion. But the number of NK cells is not directly proportional to their activity. Even when NK cells appear to be ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCC12N5/0646A61K35/17C12N2500/34C12N2501/53C12N2501/599
Inventor 王晓冰
Owner 王晓冰
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