In-vitro amplification culture method of NK cells

A technology of NK cells and a culture method, applied in the field of cell culture, can solve the problems of side effects, insufficient therapeutic effect, insufficient satisfaction, etc., and achieve the effects of strong safety performance, simple and feasible cultivation method, and improved proliferation rate and purity.

Inactive Publication Date: 2020-01-14
CHENGDU EXAB BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, those methods use cultured cancer cells and gene transfer cells in order to strengthen NK cells, and there are unresolved problems in terms of safety and practicality in clinical application.
In addition, NK cell growth efficiency, cell activity, and purity are not yet at a sufficiently satisfactory level.
[0011] As mentioned above, any of the conventional immunotherapy methods has problems that cannot obtain sufficient therapeutic effects, are accompanied by serious side effects, or other problems that should be improved

Method used

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  • In-vitro amplification culture method of NK cells
  • In-vitro amplification culture method of NK cells
  • In-vitro amplification culture method of NK cells

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0055] (2) Preparation of blood

[0056] When the blood used in this step is blood collected directly from a living body, the collection method may follow a known blood collection method. For example, peripheral blood can be collected by injecting it into a peripheral vein, etc.; bone marrow fluid can be collected by bone marrow aspiration; Hereinafter, the collection of peripheral blood will be specifically described with an example.

[0057] Peripheral whole blood can be collected by inserting an injection needle into a peripheral blood vessel of a living body, for example, a vein or an artery, and using a known whole blood collection method such as a vacuum blood collection tube or a blood collection bag. The volume to be taken varies depending on the amount of the necessary NK cell blood preparation, for example, in the case of manufacturing the aforementioned blood preparation to be administered once per adult, usually 40 mL to 60 mL is sufficient. However, when the num...

Embodiment 1

[0084] A method for in vitro expansion and culture of NK cells, comprising the following steps:

[0085] (1) Preparation of autologous plasma

[0086] 10 mL of peripheral whole blood was collected from the donor's vein using a heparin anticoagulated blood collection tube. The collected peripheral whole blood was transferred to a sterile conical centrifuge tube, centrifuged at 900 g for 10 minutes, and the supernatant was separated as plasma. To the remaining blood cell components after plasma collection, sterile PBS was added in an amount twice that of the whole blood before plasma separation to make a blood cell solution for the subsequent separation of PBMCs. The plasma was inactivated by treatment at 56° C. for 30 minutes, and centrifuged at 900 g for 10 minutes to remove platelets and the like. Thereafter, the plasma was stored at 4°C. This plasma was used as autologous plasma for cell culture added to the medium, and the necessary amount was used every time the medium ...

Embodiment 2

[0105] A method for in vitro expansion and culture of NK cells, comprising the following steps:

[0106] (1) Preparation of autologous plasma

[0107] 20 mL of peripheral whole blood was collected from the donor's vein using a heparin anticoagulated blood collection tube. The collected peripheral whole blood was transferred to a sterile conical centrifuge tube, centrifuged at 900 g for 10 minutes, and the supernatant was separated as plasma. To the remaining blood cell components after plasma collection, 2 times the amount of sterile PBS compared to the whole blood volume before plasma separation was added to make a blood cell solution for the subsequent separation of PBMCs. The plasma was inactivated by treatment at 56° C. for 30 minutes, and then centrifuged at 900 g for 10 minutes to remove platelets and the like. Thereafter, plasma was stored at 4°C. This plasma was used as autologous plasma for cell culture added to the medium, and the necessary amount was used every t...

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Abstract

The invention discloses an in-vitro amplification culture method of NK cell bodies. The method comprises the following steps: (1) adding a stimulating factor and a proportion regulator with a final concentration of 0.1-10nM into treated blood containing NK cells, and culturing for 3-4 days; and (2) adjusting the cell density to 0.5-1.5*10 < 6 > cells/mL, continuously adding a final proportion regulator and cell factors, and continuously culturing for 20-30 days. In the process of culturing the NK cells, allogeneic cells do not exist, the related factors are factors harmless to a human body inthe clinical level, the safety performance is high, the phenotype and number of the cultured cells are stable, and the culture method is simple, convenient and feasible.

Description

technical field [0001] The invention belongs to the technical field of cell cultivation, in particular to a method for in vitro expansion and cultivation of NK cells. Background technique [0002] With the advancement of medicine, the survival rate of cancer patients has been significantly improved, but it is still a refractory disease. The standard methods for cancer treatment are surgery, chemotherapy, and radiation therapy. In recent years, immunotherapy has attracted attention as a new treatment method, and various methods have been developed so far. Immunotherapy refers to a method of treating cancer, viral infection, etc. by using the body's immune system. Such as cytokine therapy, cellular immunotherapy. [0003] Cytokine therapy refers to a method of killing cancer cells and virus-infected cells by directly administering cytokines capable of proliferating or activating lymphocytes such as T cells and NK cells into a living body. For example, there are treatments b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/06C12N2501/21C12N2501/2302C12N2501/2318C12N2501/25C12N2501/599
Inventor 张志新卓越张勇杨飞
Owner CHENGDU EXAB BIOTECH CO LTD
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