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NK cell culture system and application thereof

A technology of NK cells and culture system, applied in cell culture active agents, cell culture supports/coatings, animal cells, etc., can solve the problems of cumbersome operation and high cost of the culture process, and achieve a safe preparation method, low cost, and low cost. therapeutic effect

Active Publication Date: 2021-06-01
GUANGZHOU DOUBLLE BIOPRODUCT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The described NK cell culture process is relatively cumbersome and costly

Method used

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  • NK cell culture system and application thereof
  • NK cell culture system and application thereof
  • NK cell culture system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 NK cell culture system

[0037]1. Coating antibody: 96.5% PBS, 0.5% anti-CD3 mAb, 1% anti-CD137 mAb, 0.5% anti-CD161 mAb, 1% anti-CD16 mAb, 0.25% anti-HER2 mAb and 0.25% anti-PD -L1 mAb.

[0038] 2. NK cell activating factor: 98.25% PBS, 1% IL-2, 0.02% IL-7, 0.02% IL-9, 0.03% IL-12, 0.05% IL-15, 0.5% IL-18, 0.03% IL -21, 0.05% IFN-alpha and 0.05% IFN-lamda.

[0039] 3. NK cell basal medium: 99.5% serum-free medium (RPMI1640), 0.1% vitamin mixture (A / B / C / D / E), 0.2% polysaccharide sulfate and 0.2% tea active polysaccharide.

[0040] 4. NK cell activation medium contains: 89.488% NK cell basal medium, 0.01% lipopolysaccharide, 0.002% resveratrol, 0.5% NK cell activating factor and 10% autoinactivated plasma.

[0041] 5. NK cell expansion medium: NK cell basal medium and 500IU / mL IL-2.

Embodiment 2

[0042] In vitro culture expansion of embodiment 2 NK cells

[0043] 1. Antibody coating: After diluting the coated antibody 1:5 with normal saline, add it to a T75 cell culture flask. After standing overnight at 4°C or incubating at room temperature for 2 hours, slowly decant to remove the supernatant, take 10 mL of normal saline to wash the culture bottle once, slowly decant the normal saline before use.

[0044] 2. Autologous Plasma Preparation and Peripheral Blood Mononuclear Cell Isolation

[0045] 1) Centrifuge the anticoagulated whole blood at 2000rpm at room temperature for 10min. Take the supernatant, and place the supernatant in a 56°C water bath for 30 minutes to inactivate. Then, centrifuge at 3500 rpm for 10 min at room temperature. The supernatant was taken as inactivated autologous plasma.

[0046] 2) After diluting the lower blood layer with 0.9% sodium chloride injection 1:1, add it to the top of the lymphatic separation liquid, the ratio of the diluent to ...

experiment example 1

[0057] Experimental example 1 Cell number and activity detection

[0058] The cell suspension prepared in Example 2 and Comparative Example 1 was mixed with 0.2-0.4% trypan blue solution 1:1, and counted by countstar automatic cell counter. The test results are shown in Table 1 below.

[0059] Table 1

[0060] Test items Survival rate cell number Example 2 93％ 6.04×10 9

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Abstract

The invention provides an NK cell culture system and an application thereof, and belongs to the technical field of immune cell therapy. The NK cell reinfusion solution is prepared through the steps of peripheral blood mononuclear cell separation, cell activation, proliferation and the like by utilizing the NK cell culture system, exogenous animal serum does not need to be added, exogenous trophoblast cells do not need to be adopted either, and the preparation method is safer, easy to operate, low in cost, and suitable for clinical-grade production of the NK cells. The tumor killing experiments show that the NK cell culture system can relieve NK cell inhibition to a greater extent, activate NK cells and improve the tumor killing activity of the NK cells.

Description

technical field [0001] The invention relates to the technical field of immune cell therapy, in particular to an NK cell culture system and its application. Background technique [0002] Tumor is a disease with high mortality rate, easy recurrence and easy metastasis. In recent years, studies have found that the occurrence and development of tumors and postoperative tumor metastasis are closely related to the suppression of the body's immune function. Therefore, the use of immunological methods to inhibit the occurrence and development of tumors, reduce and prevent postoperative metastasis has become a hot spot in anti-tumor research. In 2013, the American Journal of Science announced that the era of tumor immunotherapy is coming. Tumor immunotherapy is to stimulate or mobilize the immune function of the body and enhance the anti-tumor immunity of the tumor microenvironment, so as to achieve the purpose of controlling and killing tumor cells. Lymphokine-activated killer cel...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00A61P35/02
CPCA61K35/17A61P35/00A61P35/02C12N5/0646C12N2500/38C12N2501/11C12N2501/2302C12N2501/2307C12N2501/2309C12N2501/2312C12N2501/2315C12N2501/2318C12N2501/2321C12N2501/24C12N2501/51C12N2501/515C12N2501/59C12N2501/599C12N2501/90C12N2501/999C12N2533/50
Inventor 黄文林周晓鸿田烁
Owner GUANGZHOU DOUBLLE BIOPRODUCT CO LTD
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