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Rapid amplification method of natural killer cells

A natural killer cell, fast technology, applied in the direction of animal cells, vertebrate cells, cell culture active agent, etc., can solve the problems of complex culture process, large amount of culture medium usage, poor cell effect, etc.

Active Publication Date: 2018-05-01
北京汇智驰康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method for rapid expansion of natural killer cells, which is used to solve the problems of poor effect of cells cultured by existing culture methods, complicated culture process, and large amount of culture fluid used

Method used

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  • Rapid amplification method of natural killer cells
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  • Rapid amplification method of natural killer cells

Examples

Experimental program
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Effect test

Embodiment 1

[0035] The natural killer cell rapid expansion method of the present embodiment comprises the following steps:

[0036] Step A: Preparation of natural killer cell medium, the prepared medium includes: complete activation medium and cell expansion medium; complete activation medium includes: IL-12 with a concentration of 800U / ml, IL-12 with a concentration of 800U / ml 15,800 U / ml IFN-γ in DMEM medium; cell expansion medium includes: RPMI-1640 medium with a concentration of 800 U / ml IL-2 and 800 U / ml IL-15. The consumption of complete activation medium is 40ml; the consumption of cell expansion medium is 800ml.

[0037] Step B: Isolation and culture of natural killer cells;

[0038] Step B1: Transfer 50ml of anticoagulated peripheral blood to a 50ml centrifuge tube, centrifuge at 700g, absorb the upper layer of plasma into another centrifuge tube, and set aside;

[0039] Step B2: Dilute the plasma with physiological saline 0.5 times the volume of the plasma to obtain diluted bl...

Embodiment 2

[0045] The natural killer cell rapid expansion method of the present embodiment comprises the following steps:

[0046] Step A: Preparation of natural killer cell medium, the prepared medium includes: complete activation medium and cell expansion medium; complete activation medium includes: IL-12 at a concentration of 1000U / ml, IL-12 at a concentration of 1000U / ml 15. 1000 U / ml IFN-γ DMEM medium; cell expansion medium includes: 1000 U / ml IL-2, 1000 U / ml IL-15 RPMI-1640 medium. The consumption of complete activation medium is 45ml; the consumption of cell expansion medium is 1000ml.

[0047] Step B: Isolation and culture of natural killer cells;

[0048] Step B1: Transfer 80ml of anticoagulated peripheral blood to a 50ml centrifuge tube, centrifuge at 700g, absorb the upper layer of plasma into another centrifuge tube, and set aside;

[0049] Step B2: Dilute the plasma with normal saline that is 1 times the volume of the plasma to obtain diluted blood;

[0050] Step B3: sepa...

Embodiment 3

[0055] The natural killer cell rapid expansion method of the present embodiment comprises the following steps:

[0056] Step A: Preparation of natural killer cell medium, the prepared medium includes: complete activation medium and cell expansion medium; complete activation medium includes: IL-12 at a concentration of 1200U / ml, IL-12 at a concentration of 1200U / ml 15, 1200U / ml IFN-γ DMEM medium; cell expansion medium includes: 1200U / ml IL-2, 1200U / ml IL-15 RPMI-1640 medium. The consumption of complete activation medium is 50ml; the consumption of cell expansion medium is 1200ml.

[0057] Step B: Isolation and culture of natural killer cells;

[0058] Step B1: Transfer 100ml of anticoagulated peripheral blood to a 50ml centrifuge tube, centrifuge at 700g, absorb the upper layer of plasma into another centrifuge tube, and set aside;

[0059] Step B2: Dilute the plasma with physiological saline twice the volume of the plasma to obtain diluted blood;

[0060] Step B3: separatin...

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Abstract

The invention provides a rapid amplification method of natural killer cells. The rapid amplification method of the natural killer cells comprises the following steps: A, preparing a culture medium ofthe natural killer cells; B, carrying out isolation and culture of the natural killer cells; B1, removing anticoagulant peripheral blood into a centrifuge tube, performing centrifugation, absorbing upper-layer plasma into another centrifuge tube for later use; B2, diluting the plasma with physiological saline of which the volume is 0.5 to 2 times that of the plasma to obtain diluted blood; B3, separating PBMCs from the diluted blood with Ficoll-Paque; B4, resuspending 3 to 5*10<7> / ml PBMCs in a 40-60 ml culture vessel of a completely activated culture medium, and placing in an incubator of which the temperature is 35 to 40 DEG C and the CO2 concentration is 2 to 8% to be cultured for 2 to 4 days; B5, supplementing a cell expansion culture medium in the completely activated culture medium,and continuously carrying out culture to obtain the natural killer cells. The culture method of the invention can amplify a large number of the natural killer cells in a short time, and is lower in amount of the culture medium, and simple, convenient and safe in culture.

Description

technical field [0001] The invention relates to cell culture technology, in particular to a method for rapidly expanding human natural killer cells. Background technique [0002] Natural killer cells (natural killer cells, referred to as natural killer cells) were discovered by Herberman et al. Since natural killer cells express many lymphocyte surface markers and are derived from lymphoid progenitor cells in the bone marrow, they are classified as lymphocytes. Natural killer cells belong to a subgroup of white blood cells, which can kill target cells without antigen stimulation, and can exert anti-tumor functions through the functions of immune clearance and immune surveillance. [0003] The mechanism of natural killer cells to clear tumor cells can be divided into three categories: one is to kill tumor cells by releasing cytotoxic particles; the other is to kill tumor cells by activating the tumor cell apoptosis system through the protein synthesized on the cell surface; ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/125C12N2501/2302C12N2501/2312C12N2501/2315C12N2501/24C12N2501/515C12N2501/599
Inventor 段海峰
Owner 北京汇智驰康生物科技有限公司
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