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Method for abundantly amplifying NK (natural killer) cells from mononuclear cell of peripheral blood

A technology of NK cells and nuclear cells, applied in blood/immune system cells, animal cells, vertebrate cells, etc., can solve problems such as troublesome safety, high cost and insufficient amplification methods, and achieve the effect of improving killing ability

Active Publication Date: 2017-08-08
SHANGHAI LIFE SCI & TECH CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] In order to solve the problems of insufficient NK cell purity, low amplification factor, high cost and troublesome expansion method and insufficient safety in the prior art, the present invention provides a method for amplifying NK cells in large quantities from peripheral blood mononuclear cells. The method, which does not contain animal serum and tumor cell components, thus greatly improves the safety and reliability of NK cells, and has a good application prospect

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  • Method for abundantly amplifying NK (natural killer) cells from mononuclear cell of peripheral blood
  • Method for abundantly amplifying NK (natural killer) cells from mononuclear cell of peripheral blood
  • Method for abundantly amplifying NK (natural killer) cells from mononuclear cell of peripheral blood

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Embodiment 1

[0031] 1. Isolation process of PBMC

[0032]Dissolve 500 μg of human CD16 monoclonal antibody in 25 mL of normal saline, and then completely add it into a T175 culture flask. Make the liquid cover the bottom of the bottle, and place the culture bottle flat in a refrigerator at 4°C to coat overnight (16 hours).

[0033] Separate the peripheral blood from the peripheral blood of the patient, suck out the blood with a syringe and slowly drop it into a 50ml centrifuge tube. Centrifuge at a speed of 600g for 20 minutes; the upper layer of the separated blood is a plasma layer, and the lower layer is a blood cell layer. Aspirate the upper layer of plasma, seal it, inactivate at 56°C for 30 minutes, and complete the inactivation; place at 4°C for 15 minutes, and centrifuge at 1800g for 15 minutes. Store at -20°C or 4°C. Resuspend the lower blood cell pellet with the same volume of normal saline as the blood cell pellet, and mix well.

[0034] The human lymphocyte separation solut...

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Abstract

The invention relates to a method for abundantly amplifying NK (Natural Killer) cells from a mononuclear cell of peripheral blood. The method comprises the following steps of collecting the mononuclear cell of the peripheral blood, first putting the mononuclear cell into a culture bottle enveloped by a CD16 monoclonal antibody, culturing the mononuclear cell, and adding IL-2, IL-15, OK432 and inactivated autoserum into a culture medium; subsequently, transferring the cell into a culture bottle which is not enveloped by the CD16 monoclonal antibody, culturing the cell, adding the IL-2, the IL-15 and the inactivated autoserum into the culture medium; afterwards, transferring the cell into a culture bag, culturing the cell, and adding IL-4 into the culture medium on the last third day, and continuously culturing the cell, so as to obtain abundant NK cells. According to the method, components of animal serum and a tumor cell are not contained; therefore, the safety and the reliability of the NK cells are improved to a great extent; the method has a favorable application prospect.

Description

technical field [0001] The invention belongs to the field of cell differentiation and culture, in particular to a method for massively expanding NK cells from peripheral blood mononuclear cells. Background technique [0002] Natural killer cells (natural killer, NK) are derived from bone marrow lymphoid stem cells. Their differentiation and development depend on the microenvironment of bone marrow or thymus. They are mainly distributed in peripheral blood and spleen, and also exist in a small amount in lymph nodes and other tissues. NK cells do not express specific antigen recognition receptors, and are the third type of lymphocytes different from T and B lymphocytes. NK cells express CD56 but lack CD3 and are usually defined by the marker combination CD3-CD56+. NK cells mainly exist in peripheral blood and spleen, accounting for about 5% to 15% of peripheral blood lymphocytes. [0003] NK cells can directly kill tumor cells and infected cells without antigen pre-sensitiza...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 李新峰朱灏张钰周雁冰
Owner SHANGHAI LIFE SCI & TECH CO LTD
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