Method for in vitro multiplication culture of NK cells

An in vitro culture and NK cell technology, applied in the field of in vitro culture and expansion of NK cells, can solve the problems of instability, low amplification multiple and purity, and unverified safety, and achieve high safety and strong expansion ability. , the effect of simple amplification method

Active Publication Date: 2019-04-16
福建省海西细胞生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The safety of clinical application of the first two methods of expanding NK cells has not been verified, and K562 cells have certain risks as tumor cells, so these two methods are mostly used in scientific r

Method used

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  • Method for in vitro multiplication culture of NK cells
  • Method for in vitro multiplication culture of NK cells
  • Method for in vitro multiplication culture of NK cells

Examples

Experimental program
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Effect test

Embodiment 1

[0024] A method for culturing NK cells in vitro, after culture bottle Coating, peripheral blood collection, plasma inactivation, mononuclear cell separation, seed bottle, amplification, bottle transfer, cell harvesting and detection, the specific steps are as follows:

[0025] (1) Culture flask Coating: Prepare a mixed solution of heparin sodium, CD16 and human immunoglobulin, in which the content of heparin sodium is 500U / ml, the content of CD16 is 5ug / ml, and the content of human immunoglobulin is 1mg / ml. 5ml covered the bottom of non-TC-treated T75 culture flask, and placed the culture flask in a refrigerator at 4°C. After 12 hours, the liquid in the culture bottle was discarded, and the culture bottle was washed twice with PBS for later use.

[0026] (2) Peripheral blood collection: Volunteers tested negative for hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody, Treponema pallidum antibody, and cytomegalovirus IgM antibody. Take 50m...

Embodiment 2

[0034] (1) Coating of culture flasks: Cover the bottom of non-TC-treated T75 culture flasks with a coating solution containing sodium heparin, CD16 and human immunoglobulin, and place the culture flasks in a refrigerator at 4°C. After 10 hours, the liquid in the culture bottle was discarded, and the culture bottle was washed twice with PBS for the following operations. The content of heparin sodium in the coating solution is 500U / ml, the content of CD16 is 5ug / ml, and the content of human immunoglobulin is 1mg / ml.

[0035] (2) Peripheral blood collection: Volunteers tested negative for hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody, Treponema pallidum antibody, and cytomegalovirus IgM antibody. Take 50ml of peripheral blood, put it in a 10ml sterile sodium heparin blood collection tube, and separate it within 2 hours.

[0036] (3) Plasma inactivation: Put the peripheral blood into a centrifuge and centrifuge at 3000 rpm for 10 minutes...

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Abstract

The invention relates to a method for in vitro multiplication culture of NK cells. The method comprises coating a culture bottle with heparin sodium, CD16 and human immunoglobulin, collecting peripheral blood, inactivating plasma, separating mononuclear cells, using a serum-free medium (containing autologous plasma, IL-2, IL-15 and IL-18) to culture multiplication cells, harvesting and detecting the cells and the like. Trophoblast cells or magnetic beads are not used for sorting and purifying, and the method has simple operation, high safety, good amplification effect and high clinical application value.

Description

technical field [0001] The invention relates to the technical field of human cell culture, in particular to a method for in vitro culture and expansion of NK cells. Background technique [0002] NK cells, also known as natural killer cells, are an important natural immune system lymphocyte in the human body. Its phenotype is generally CD3 - CD16 + CD56 + . The advantage of NK cells is that they can recognize and kill tumor cells without antigen sensitization and MHC restriction, and play an important role in the body's anti-tumor and early anti-virus immune responses. Therefore, NK cells have great prospects for the treatment of tumor patients, but the low proportion and quantity of NK cells in the body limit the application of NK cells, so the technology of expanding NK cells in vitro has begun to develop. [0003] At present, there are three main technologies for expanding NK cells in vitro: one is to use immunomagnetic bead cell sorting to obtain purified NK cells fr...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/84C12N2501/599C12N2501/2315C12N2501/2318C12N2501/2302
Inventor 常满倩檀东安沈骧一黄凤杰
Owner 福建省海西细胞生物工程有限公司
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