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Method for in vitro activating and amplifying natural killer cells

A cell and nuclear cell technology, applied in the field of biomedicine, can solve the problem of low overall process cost of NK cells, and achieve the effects of reducing the cost of in vitro expansion and culture, increasing the ratio and expansion multiple, large market prospects and economic value

Active Publication Date: 2018-05-08
深圳市赛欧细胞技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For this reason, the technical problem to be solved by the present invention is to overcome the deficiencies in the prior art. After a large number of experimental studies such as screening of inducing activators, especially the in vitro activation culture and expansion technology of NK cells have been optimized, thus finding a Easy to operate and economical and effective NK cell culture and expansion technology, featuring high NK cell yield, good growth status, high killing activity, and low overall process cost

Method used

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  • Method for in vitro activating and amplifying natural killer cells
  • Method for in vitro activating and amplifying natural killer cells
  • Method for in vitro activating and amplifying natural killer cells

Examples

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Embodiment 1

[0044] Example 1 This example discloses a method for in vitro culture and expansion of natural killer cells (NK cells). The method (taking 50ml of human peripheral blood as an example) is as follows:

[0045] A. Activator Pretreatment

[0046] 1) Draw 500ul of activator, containing hyaluronic acid (concentration range between 0.10mg-10.00mg / ml) and CD16 antibody (concentration range between 0.5μg-2μg / ml), add to a 175cm 2 in a culture bottle. Add 20ml of PBS and mix thoroughly to disperse the liquid at the bottom of the bottle;

[0047] 2) Activate at 4°C for 12-16 hours; or store in a refrigerator at 4°C for 2 weeks.

[0048] 3) Aspirate off the activator, add physiological saline along the side wall of the culture bottle, shake the culture bottle gently to fully disperse the liquid.

[0049] 4) Aspirate off the normal saline, and the washed culture bottle should be used immediately.

[0050] B. Mononuclear cell isolation, plasma collection and processing

[0051] 1) Tur...

Embodiment 2

[0075] Example 2 This example discloses a method for in vitro culture and expansion of natural killer cells (NK cells). The method (taking 50ml of human umbilical cord blood as an example) is as follows:

[0076] A. Activator Pretreatment

[0077] 1) Draw 500u l of activator, containing hyaluronic acid (concentration range between 0.10mg-10.00mg / ml) and CD16 antibody (concentration range between 0.5μg-2μg / ml), add to a 175cm 2 in a culture bottle. Add 20ml of PBS and mix thoroughly to disperse the liquid at the bottom of the bottle.

[0078] 2) Activate at 4°C for 12-16 hours; or store in a refrigerator at 4°C for 2 weeks.

[0079] 3) Aspirate off the activator, add physiological saline along the side wall of the culture bottle, shake the culture bottle gently to fully disperse the liquid.

[0080] 4) Aspirate off the normal saline, and the washed culture bottle should be used immediately.

[0081] B. Mononuclear cell isolation, plasma collection and processing

[0082] 1...

experiment example 1

[0107] Experimental Example 1 The NK cells obtained in Example 1 were subjected to microscopic observation at the stage of induction and activation, and the results were as follows: figure 1 Significant NK cell colony formation is shown.

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to a method for in vitro activating and amplifying natural killer cells. The method comprises the steps that in activator pretreatment, an activator and PBS are added into a culture flask and mixed, wherein the activator contains 0.10-100.00 mg / ml of hyaluronic acid and 0.1-20 microgram / ml of a CD16 antibody, and the volume ratio of the activator to the PBS is 1:40; activating treatment is conducted, the activator is removed, normal saline is added, the culture flask is shaken slightly, the normal saline is removed finally, and a coasted culture flask is obtained. A granulocyte-macrophage colony stimulating factor (GM-CSF) is added in a culture medium when a mononuclear cell is cultured, and the concentration is made to range from 0.05 microgram / ml to 1.00 microgram / m. Compared with an existing patented method, the method is simpler and more effective, and the in vitro amplifying and culture cost of theNK cells is effectively lowered. In the coating step, hyaluronic acid is added, and the proportion and amplification multiple of the NK cells are significantly increased; and the method has the greatmarket prospect and economic value.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to an in vitro culture and expansion method of natural killer cells (NK cells). Background technique [0002] Usually, the application process of NK cells starts from the collection of blood from different sources, and has gone through the isolation of mononuclear cells from blood (such as peripheral blood, umbilical cord blood, bone marrow), seed bottle coating, activator stimulation, specific antibody induction, Seven stages including large-scale expansion culture, NK cell harvesting, and quality control, involving mononuclear cell isolation technology, NK cell induction and activation technology in vitro, NK cell high-efficiency expansion technology in vitro, NK cell harvesting technology, NK Cell quality control technology and other key process technologies. Among them, the in vitro induction and large-scale high-efficiency expansion technology of NK cells is the basis and key step o...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/22C12N2501/2302C12N2501/2315C12N2501/599C12N2501/905
Inventor 张涤平郭子宽葛四海钱开诚刘家权
Owner 深圳市赛欧细胞技术有限公司
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