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Amplification method for NK cell without trophocyte

A technology of NK cells and trophoblasts, applied in animal cells, vertebrate cells, cell culture active agents, etc., can solve problems such as difficulty in ensuring sustainability, achieve increased quantity and purity, high amplification efficiency, and cost savings Effect

Active Publication Date: 2017-12-12
山东省齐鲁细胞治疗工程技术有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are also reports about some researchers using tumor cell lines as feeder cells to proliferate NK cells. Although the addition of feeder cells can greatly expand NK cells without senescence, its There are still many problems in clinical application. For example, after NK cells are continuously cultured in vitro for several weeks, the trophoblasts need to be removed in advance to ensure that there are no residues before entering the human body. After losing the optimal growth conditions, the cytotoxicity of NK It may decrease, and the sustainability after infusion in vivo is difficult to guarantee; adding allogeneic cells as trophoblast cells, especially the K562 cancer cell line, also has major risks and ethical issues in clinical application

Method used

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  • Amplification method for NK cell without trophocyte
  • Amplification method for NK cell without trophocyte
  • Amplification method for NK cell without trophocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Isolation of mononuclear cells from peripheral blood or umbilical cord blood

[0040] 1) In the biological safety cabinet, divide 30mL blood into 50ml centrifuge tubes, and centrifuge at 500g for 10min;

[0041] 2) After centrifugation, put the upper plasma layer into a new 50ml centrifuge tube, inactivate it in a water bath at 56°C for 30 minutes, centrifuge it at 2000g for 10 minutes, and save the supernatant for later use.

[0042] 3) Dilute the blood cells with 15mL normal saline at a ratio of 1:1, take 15mL of lymphocyte separation medium, and slowly add the mixed blood cells into the centrifuge tube containing lymphocyte separation medium at a ratio of 2:1 to make it appear Clear interface, centrifuge at 400g for 20min;

[0043] 4) After centrifugation, the liquid surface will be divided into 4 layers. From the bottom, there are red blood cells, lymphatic separation liquid layer, buffy coat layer and supernatant layer. Discard the supernatant layer, and...

Embodiment 2

[0046] Example 2 Cytokine combination optimization experiment

[0047] Cytokine combinations were divided into 3 groups: (1) autologous plasma medium group (on the basis of GT-T551H3 culture medium, add 1-10% autologous plasma, 200IU / mL IL-2); (2) co-culture Liquid group (on the basis of GT-T551H3 culture medium, add 1-10% autologous plasma, 200IU / mL IL-2, 50ng / mL IL-15 and 25ng / mL IL-21); (3) total After 7 days of culture in the mixed culture medium, it was replaced with the autologous plasma medium group.

[0048] On the 0th day of culture, the mononuclear cells obtained in Example 1 were respectively suspended in the corresponding culture medium, and the cell density was adjusted to 1.5×10 6 / mL, seeded in a 6-well plate, the final volume of each well is 2ml, the autologous plasma concentration is 5%, at 37°C, 5% CO 2 and saturated steam incubator.

[0049] On the third day of culture, add 1ml of the corresponding culture solution, the concentration of autologous plasma ...

Embodiment 3

[0055] The mass expansion culture of embodiment 3 NK cells

[0056] 1) Activation and stimulation of NK cells: Mononuclear cells were mixed with culture medium (on the basis of GT-T551H3 culture medium, containing 5% autologous plasma, 200IU / mL IL-2, 50ng / mL IL-15, 25ng / mL IL-21) was resuspended, and the cell density was adjusted to 1.5×10 6 / mL, after mixing, take 4mL and add it to a T25 culture flask. 2 and saturated water vapor incubator, add 2mL blended culture solution on the 3rd day, continue to culture for 7 days, and observe the cell morphology under the microscope.

[0057] 2) NK cell expansion culture: After 7 days of activation culture, the natural killer cells were pipetted with a 10mL pipette, then pipetted into a T75 cell culture bottle, and 8mL of fresh medium (based on the GT-T551H3 culture medium, containing 1% autologous plasma, 200IU / mL IL-2), 37℃, 5%CO 2 and saturated water vapor incubator for 2 days, and then supplemented every 2 to 3 days to maintain t...

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Abstract

The invention discloses an amplification method for an NK cell without trophocyte. The method comprises the following steps: adopting a blending culture solution for culturing the NK cell and culturing for 7 days, and then using a self-plasma culture medium for culturing, supplying the fluid per 2-3 days in the culturing process, maintaining the cell density of 2.0*106 / mL and culturing for more than 20 days. The blending culture solution is composed of 1-10% of self-plasma, 200-1500 IU / mL IL-2, 10-50ng / mL IL-15 and 10-50ng / mL IL-21 added into a lymphocyte culture medium. The self-plasma culture medium is composed of 1-10% of self-plasma and 200 IU / mL IL-2 added into a serum-free medium. According to the amplification method for the NK cell without trophocyte disclosed by the invention, cord blood or autologous peripheral blood is taken as a cell source, no trophocyte is required in the culturing process, the amplification efficiency is high and the purity of the acquired NK cell is high. The NK cell acquired according to the method can be used as a medicine compound for cell immunization therapy and is mainly used for treating and / or preventing infectious diseases and / or cancer.

Description

technical field [0001] The invention relates to a method for expanding NK cells without trophoblasts. Background technique [0002] Natural killer cells (NK cells) belong to the body's innate immune cells, which are closely related to the body's resistance to malignant tumors, viral infections and immune regulation. Its immunophenotype is mainly characterized by CD3- / CD56+, which is a lymphocyte subset that does not express T-cell receptors or B-cell receptors. Its killing of tumor cells or infected cells does not depend on the participation of antibodies, nor does it require antigen stimulation and sensitization; the recognition of target cells is not limited by the major histocompatibility complex (MHC), and can directly interact with target cells Release perforin and granzyme by contact to kill target cells; NK cytotoxic factor can also be released to bind to NK cytotoxic factor (NK cytotoxic factor, NKCF) receptors of target cells, selectively killing and lysing target ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2302C12N2501/2315C12N2501/2321
Inventor 孔群芳李悦谭毅梁晨张慧慧
Owner 山东省齐鲁细胞治疗工程技术有限公司
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