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Kit for culturing natural killer cells in vitro and use method and application thereof

A natural killer cell, in vitro culture technology, used in cell culture active agents, animal cells, culture processes, etc., can solve problems such as poor quantity and purity

Pending Publication Date: 2020-10-30
湖南玖森生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the obtained culture products, NK cells only expanded by 102.81±94.06 times, and the traditional Ficoll density gradient centrifugation method was used to separate NK cells, and the quantity and purity were not good

Method used

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  • Kit for culturing natural killer cells in vitro and use method and application thereof
  • Kit for culturing natural killer cells in vitro and use method and application thereof
  • Kit for culturing natural killer cells in vitro and use method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] This embodiment provides a kit for culturing natural killer cells in vitro, which specifically includes:

[0081] 1. 10mL coating solution: DPBS (Duchenne's phosphate buffered saline) containing recombinant human fibronectin at a final concentration of 10 μg / mL and humanized anti-human CD3 monoclonal antibody at 10 μg / mL;

[0082] 2. 50 mL incubation solution: DPBS containing inactivated and filtered fetal bovine serum with a volume ratio of 20% and humanized anti-human CD3 monoclonal antibody at 2.5 μg / mL;

[0083] 3. 60 mL serum-free medium 1: In Alys-505N medium, add IL-2 at a final concentration of 1200 U / mL, IL-15 at a final concentration of 10 μg / mL, IL-18 at 10 μg / mL, and 10 μg / mL mL of IL-21 and autologous plasma at a final concentration of 10%;

[0084] 4. 140mL serum-free medium 2: In Alys-505N medium, add IL-2 at a final concentration of 1000U / mL, IL-15 at a final concentration of 5μg / mL, IL-18 at 5μg / mL, and 5μg / mL mL of IL-21 and autologous plasma at a fi...

Embodiment 2

[0088] This embodiment provides a kit for culturing natural killer cells in vitro, which specifically includes:

[0089] 1. 10mL coating solution: DPBS (Duchenne's phosphate buffer saline) containing recombinant human fibronectin with a final concentration of 8 μg / mL and humanized anti-human CD3 monoclonal antibody at 8 μg / mL;

[0090] 2. 50mL incubation solution: DPBS containing inactivated and filtered fetal bovine serum with a volume ratio of 10% and humanized anti-human CD3 monoclonal antibody at 2 μg / mL;

[0091] 3. 60 mL serum-free medium 1: Add IL-2 at a final concentration of 1000 U / mL, IL-15 at a final concentration of 8 μg / mL, IL-18 at 8 μg / mL, and IL-18 at a final concentration of 8 μg / mL in GT T551 medium IL-21 and autologous plasma with a final concentration of 8%;

[0092] 4. 140mL serum-free medium 2: In GT T551 medium, add IL-2 at a final concentration of 800U / mL, IL-15 at a final concentration of 4μg / mL, IL-18 at 4μg / mL, and IL-18 at a final concentration of ...

Embodiment 3

[0096] This embodiment provides a kit for culturing natural killer cells in vitro, which specifically includes:

[0097] 1. 10mL coating solution: DPBS (Duchenne's phosphate buffered saline) containing recombinant human fibronectin at a final concentration of 12 μg / mL and humanized anti-human CD3 monoclonal antibody at 12 μg / mL;

[0098] 2. 50 mL incubation solution: DPBS containing inactivated and filtered fetal bovine serum with a volume ratio of 30% and humanized anti-human CD3 monoclonal antibody at 3 μg / mL;

[0099] 3. 60 mL serum-free medium 1: In Alys-505N medium, add IL-2 at a final concentration of 1500 U / mL, IL-15 at a final concentration of 12 μg / mL, IL-18 at 12 μg / mL, and IL-18 at a final concentration of 12 μg / mL mL of IL-21 and autologous plasma at a final concentration of 8%;

[0100] 4. 140mL serum-free medium 2: Add IL-2 with a final concentration of 1200U / mL, IL-15 with a final concentration of 6μg / mL, IL-18 with a final concentration of 6μg / mL, and IL-18 wi...

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Abstract

The invention provides a kit for culturing natural killer cells in vitro and a use method and application of the kit. The kit comprises a coating solution, an incubation solution, a serum-free culturemedium 1, a serum-free culture medium 2, a serum-free culture medium 3 and a serum-free culture medium 4, wherein the serum-free culture medium 1, the serum-free culture medium 2 and the serum-free culture medium 3 are serum-free culture media containing interleukin 2, interleukin 15, interleukin 18, interleukin 21 and autologous plasma, and the serum-free culture medium 4 is a serum-free culturemedium comprising interleukin 2, interleukin 15, interleukin 18 and interleukin 21. By reasonably matching the types and concentrations of autologous plasma and interleukin in each culture medium, high-yield and high-purity NK cells can be efficiently prepared under the conditions of fewer seed cells and no separation and purification.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to an in vitro culture method of natural killer cells, in particular to a kit for in vitro culture of natural killer cells and its use and application. Background technique [0002] Natural killer cells (NK) are important immune cells of the body. As the first line of defense of the body's defense system, they can not only exert their main tumor-killing effect in the natural immune system, but also secrete different Cytokines and various chemokines regulate the body's acquired immune response and are indispensable effector cells for the body to exert immune effects. Compared with αβT cells, γδT cells, CIK and other cells used for immunotherapy, NK cells have received more attention and attention due to their high cell killing activity, rapid onset of action, and not being restricted by MHC. Clinical application. [0003] However, the experimental results show that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/90C12N2501/2302C12N2501/2315C12N2501/2318C12N2501/2321C12N2500/84C12N2501/515C12N2533/52
Inventor 易亮梁秋彬钱春明颜春江罗国辉吴佑星陈志浩欧阳建军汪金球
Owner 湖南玖森生物科技有限公司
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