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Method for efficiently amplifying natural killer cells from peripheral blood in high purity manner

A natural killer cell and peripheral blood technology, which is applied in the field of high-efficiency and high-purity expansion of natural killer cells, can solve the problems of unresolved safety hazards, low purity, and cumbersome methods and processes, and achieves optimization of activation and amplification conditions and simplified operations. Process, enhance the effect of cytotoxicity

Active Publication Date: 2017-11-07
SHANGHAI LIFE SCI & TECH CO LTD
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Problems solved by technology

Without using trophoblast cells, the culture effect and culture purity are not ideal. Using trophoblast cells will produce NK cells with higher purity. However, K562 cells themselves are a kind of tumor cells, so their safety hazards are still not ruled out.
The method of immunomagnetic beads is cumbersome and costly, and the proliferation rate of pure NK cells is slow, and exogenous substances (magnetic beads, components in the eluate, antibodies, etc.)
Therefore, people have been trying to expand NK cells only by using pure cytokines, but the method of using only pure cytokines generally can only expand NK cells by tens of times or even only a dozen times, and the purity is not high, which is still limited. The clinical use of NK cells

Method used

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  • Method for efficiently amplifying natural killer cells from peripheral blood in high purity manner
  • Method for efficiently amplifying natural killer cells from peripheral blood in high purity manner
  • Method for efficiently amplifying natural killer cells from peripheral blood in high purity manner

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Embodiment 1

[0032] 1. Coating culture flasks with anti-CD16 and CD226 monoclonal antibodies

[0033] Dilute anti-human CD16 and CD226 monoclonal antibodies with serum-free medium, and add 4 ml of serum-free medium containing 2 μg / ml CD16 monoclonal antibody and 1 μg / ml CD226 monoclonal antibody to a T75 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).

[0034] Second, the separation process of PBMC

[0035] 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...

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Abstract

The invention relates to a method for efficiently amplifying natural killer cells from peripheral blood in a high purity manner. The method comprises the following steps: collecting 100ml-200ml of peripheral blood, separating mononuclear cell, putting the mononuclear cell into a culture bottle coated with CD16 monoclonal antibody and CD226 monoclonal antibody for culturing, and activating the cell by virtue of IFN-gamma; after the activation is carried out for 24 hours, adding IL-2, IL-15, IL-21, OK432, CD2 monoclonal antibody and inactivated autoserum into a culture medium; transferring the cell into a culture bottle which is not coated with CD16 monoclonal antibody and CD226 monoclonal antibody for culturing, and supplementing liquid to the cell once in each 2-3 days; and finally transferring the cell into a culture bag for culturing, and collecting NK cell in the 21th day. According to the method, no any heterologous serum component or trophoblast cell is used, so that the safety and reliability of the prepared NK cell are guaranteed; and the method has good clinical application values.

Description

technical field [0001] The invention belongs to the field of cell differentiation and culture, and in particular relates to a method for amplifying natural killer cells with high efficiency and high purity from peripheral blood. Background technique [0002] Natural killer cells (natural killer, NK) are important innate immune cells of the body, differentiated from bone marrow hematopoietic stem cells, and their development and maturation depend on the bone marrow or thymus microenvironment; mature NK cells are mainly distributed in peripheral blood and spleen, and in lymph nodes and other organizations also exist in small quantities. NK cells are larger and contain cytoplasmic granules, so they are called large granular lymphocytes. Because they do not express specific antigen recognition receptors, NK cells are the third type of lymphocytes different from T and B lymphocytes. NK cells express CD56 and CD16, but lack CD3, usually with marker combination CD3 - CD56 + to i...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 李新峰周雁冰汪佳莹
Owner SHANGHAI LIFE SCI & TECH CO LTD
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