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A kind of uncoated NK cell in vitro expansion and culture method

A technology of NK cells and in vitro expansion, applied in the field of cell culture, can solve the problems of cumbersome coating operation, increase the risk of bacterial infection, and low flow results, and achieve the effects of low cost, strong stability and simple operation.

Active Publication Date: 2022-02-11
GUANGDONG XIANKANGDA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome factors such as the high cost of in vitro culture of NK cells, the use of exogenous serum, the use of feeder cells, the low flow cytometry results, the cumbersome coating operation and the increased risk of bacterial infection, etc., using IL-2, IL- 12. IL-15, IL-18, CD16 monoclonal antibody, and HER2 monoclonal antibody were stimulated to induce NK cells, autologous serum ensured nutrition, and then amplified with serum-free complete medium containing IL-2 and IL-15 to obtain A large number of high-purity NK cells

Method used

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  • A kind of uncoated NK cell in vitro expansion and culture method
  • A kind of uncoated NK cell in vitro expansion and culture method
  • A kind of uncoated NK cell in vitro expansion and culture method

Examples

Experimental program
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Effect test

Embodiment 1

[0030] An uncoated NK cell in vitro expansion kit and culture method, the specific steps are as follows:

[0031] (1) Collect peripheral blood from volunteers and obtain mononuclear cells by gradient centrifugation. Mononuclear cells: detection of hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody, Treponema pallidum antibody, cytomegalovirus IgM antibody and other items are all feminine;

[0032] (2) Separation of serum and whole blood cells: extract 50mL of blood, place it in a sterile sodium heparin blood collection tube, transfer the blood into a 50mL centrifuge tube, and obtain serum and whole blood cells after centrifugation;

[0033] (3) Inactivated serum: Serum was inactivated in a 56°C water bath for 20 minutes, then placed in a -20°C environment for quick freezing for 15 minutes, centrifuged at 1500g for 30 minutes to obtain clear serum, and stored at 2-8°C;

[0034] (4) Obtain mononuclear cells: After diluting whole blood cells...

Embodiment 2

[0039] A method for expanding and culturing non-coated NK cells in vitro, the specific steps are as follows:

[0040] (1) Collect peripheral blood from volunteers and obtain mononuclear cells by gradient centrifugation. Mononuclear cells: detection of hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody, Treponema pallidum antibody, cytomegalovirus IgM antibody and other items are all feminine;

[0041] (2) Separation of serum and whole blood cells: extract 50mL of blood, place it in a sterile sodium heparin blood collection tube, transfer the blood into a 50mL centrifuge tube, and obtain serum and whole blood cells after centrifugation;

[0042] (3) Inactivated serum: Serum was inactivated in a 56°C water bath for 20 minutes, then placed in a -20°C environment for quick freezing for 15 minutes, centrifuged at 1500g for 30 minutes to obtain clear serum, and stored at 2-8°C;

[0043] (4) Obtain mononuclear cells: After diluting whole blood c...

Embodiment 3

[0048] A method for expanding and culturing non-coated NK cells in vitro, the specific steps are as follows:

[0049] (1) Collect peripheral blood from volunteers and obtain mononuclear cells by gradient centrifugation. Mononuclear cells: detection of hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody, Treponema pallidum antibody, cytomegalovirus IgM antibody and other items are all feminine;

[0050] (2) Separation of serum and whole blood cells: extract 50mL of blood, place it in a sterile sodium heparin blood collection tube, transfer the blood into a 50mL centrifuge tube, and obtain serum and whole blood cells after centrifugation;

[0051] (3) Inactivated serum: Serum was inactivated in a 56°C water bath for 20 minutes, then placed in a -20°C environment for quick freezing for 15 minutes, centrifuged at 1500g for 30 minutes to obtain clear serum, and stored at 2-8°C;

[0052] (4) Obtain mononuclear cells: After diluting whole blood c...

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Abstract

The invention relates to the technical field of cell culture, in particular to a method for in vitro expansion and culture of uncoated NK cells. A method for in vitro expansion and culture of uncoated NK cells, comprising the following steps: (1) separating serum and whole blood cells; (2) inactivating serum; (3) obtaining mononuclear cells; (4) resuspending mononuclear cells ; (5) Induction of NK cells; (6) Expansion of NK cells. The invention does not need to be coated, the operation is simple, the NK cells can grow advantageously through the activation medium culture and the proliferation medium, the flow content and the amplification multiple of NK are relatively high, and the invention can be applied to the clinical experimental research and large-scale preparation of NK cells. .

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a non-coated NK cell expansion and culture method in vitro. Background technique [0002] As a major disease, tumor has always troubled human health. With the advancement of medicine, the cure rate and survival rate of cancer have been significantly improved, but at present, due to the complexity, diversity, variability and heterogeneity of tumors, a better treatment method cannot be found. At present, the treatment methods for cancer in the medical industry include surgical resection, chemotherapy and radiation therapy, but the damage to the human body is relatively large. The advantages of adoptive immunotherapy, such as outstanding targeting, less side effects, and simple intervention methods, have become a hot spot for research on tumor treatment today, and have been widely used in clinical practice. [0003] NK cells have become one of the outstanding cells of adoptiv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/90C12N2501/2302C12N2501/2312C12N2501/2315C12N2501/2318C12N2501/599C12N2501/51C12N2501/11
Inventor 薛卫巍谢海涛钟家炜刘元甲
Owner GUANGDONG XIANKANGDA BIOTECH CO LTD