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NK cell culture solution and culture method for multi-stage activation of NK cells

A technology of NK cells and culture methods, which is applied in the field of NK cell culture medium and multi-level activated NK cell culture, and can solve the problems of NK cell quantity not meeting clinical needs, poor killing effect of cancer cells, low purity, etc.

Active Publication Date: 2021-02-02
杜德(江门)生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention provides a kind of NK cell culture fluid and multi-stage activated NK cell culture method, solves the problem that the existing NK cell culture fluid amplifies and obtains NK cells whose quantity cannot meet the clinical requirement, and the purity is low, which can kill cancer cells. The problem of poor performance

Method used

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  • NK cell culture solution and culture method for multi-stage activation of NK cells
  • NK cell culture solution and culture method for multi-stage activation of NK cells
  • NK cell culture solution and culture method for multi-stage activation of NK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. Peripheral blood collection and isolation of mononuclear cells

[0049] 1. Select 25-year-old, male, healthy and disease-free, normal physical examination, and no abnormality in the four inspections of blood routine and virus (hepatitis B, hepatitis C, syphilis, AIDS) as the experimental sample, and the blood in the vein is drawn and collected in a 10ml tube 20IU / ml sodium heparin collection bag;

[0050] 2. After bringing the blood sample back to the laboratory, operate it in the safety cabinet immediately, take out 1ml blood sample for NK flow detection (such as figure 1 and shown in Table 2), and then start to separate the mononuclear cells in the remaining blood sample;

[0051] 3. Add the blood into a centrifuge tube containing Ficoll separation solution, centrifuge at 800g for 20 minutes, absorb the cells of the buffy coat, wash them twice by centrifugation, and the sediment is the peripheral blood mononuclear cells, and count them.

[0052] 2. Culture of perip...

Embodiment 2～ Embodiment 3

[0060] Peripheral blood collection and isolation of mononuclear cells and culture of peripheral blood NK cells are the same as in Example 1.

[0061] From figure 2 It can be seen from Table 2 that the number of NK cells obtained through the multistage culture of this embodiment is not only higher than that of the control group, but also the purity is significantly higher than that of other control group NK cells.

[0062] As can be seen from Table 3, the total number of cells, cell viability and NK cell content in the experimental group were higher, and there were significant differences compared with the control group 1-4 (P<0.01), indicating that compared with the control group 1 ~2 single-stage stimulation NK cell culture medium, multi-stage stimulation NK cell culture medium in the experimental group can effectively increase the number and content of NK cell expansion; compared with the 50ng / ml and 600ng / ml packages used in the control group Coating solution, the 300ng / m...

Embodiment 3

[0071] Example 3: NK kills cancer cells

[0072] When the NK cells are about to be harvested, prepare a certain number of four types of cancer cells in the logarithmic growth phase: K562, HEPG2, A549, HELA, all from the Shanghai Cell Bank of the Chinese Academy of Sciences;

[0073] Use the method of measuring the activity of lactate dehydrogenase (LDH) released from the cells into the medium to measure the cell damage, using Cytotoxicity LDH Assay Kit-WST, set the killing ratio of 5:1, 10:1, 20:1, and incubate for 4 hours ;

[0074] Use a microplate reader (with a 490nm filter) to measure the absorbance;

[0075] Cell damage rate (%)=[(A-B-E) / (C-D-E)]×100

[0076] A: Absorbance of E+T well - absorbance of background Blank

[0077] B: Absorbance of E low control well - Absorbance of background Blank

[0078] C: Absorbance of T high control wells-absorbance of background Blank

[0079] D: Absorbance of T high control Blank-absorbance of background Blank

[0080] E: Absorb...

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Abstract

The invention relates to the technical field of cell biology, in particular to an NK cell culture solution and a culture method for multi-stage activation of NK cells. The NK cell culture solution sequentially adopts a culture solution A, a culture solution B and a culture solution C to activate the NK cells through three stages in an NK cell culture process, and all the components are matched with one another to jointly promote the mass proliferation of the NK cells, and the yield of the NK cells is improved. Experimental data shows that the cell viability of the NK cells cultured by the NK cell culture solution can reach 99.11%, the total number of the cells is 80.75*10<8> obtained by culture of 4*10<7> mononuclear before induction, the content of the NK cells reaches 96% or more, the killing rate of the NK cells to four cancer cells is high, and the effect is obviously superior to that of a control group adopting secondary activation or adopting coating liquid with other concentrations.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a culture medium for NK cells and a culture method for multistage activation of NK cells. Background technique [0002] Peripheral blood is the blood that is distributed throughout the body and is usually donated for regeneration. Research in the past ten years has found that peripheral blood contains various cells that can rebuild the human hematopoietic and immune system, which can be used to improve immunity and treat more than 80 diseases. Therefore, peripheral blood has become an important source of immune cells, especially unrelated immune cells, and has become a very important human biological resource. [0003] Natural killer cells (NK) are important immune cells in the human blood system, and are also abundant in peripheral blood. As we all know, the first line of defense of the body's defense system is NK cells, which can secrete different cytokines and various c...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/515C12N2501/599C12N2501/998C12N2501/11C12N2501/2302C12N2501/2315Y02A50/30
Inventor 张玲洁陈爱华
Owner 杜德(江门)生物科技有限公司
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