Method used for in vitro proliferation of NK cells

A technology of NK cells and in vitro expansion, applied in the field of proliferation and activation of NK cells, can solve the problems of unsatisfactory purity and high cost, and achieve the effect of improving killing toxicity

Active Publication Date: 2014-04-30
SHANGHAI CLAISON BIOTECH
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Problems solved by technology

[0003] In order to solve the problems of unsatisfactory purity and high cost of NK cells after expansion in the prior art, the present invention provides a new method for in vitro expansion of NK cells, which can obtain a large number of safe, NK cells with high purity and strong killing activity, so that the cells can reach the quality index that can be safely and effectively used in clinical practice

Method used

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  • Method used for in vitro proliferation of NK cells
  • Method used for in vitro proliferation of NK cells
  • Method used for in vitro proliferation of NK cells

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Embodiment 1

[0026] 1. Isolation of peripheral blood mononuclear cells (PBMC) and culture of NK cells

[0027] Add the coating solution containing anti-CD3 and CD16 antibodies to the cell culture flask in advance, incubate at room temperature for 1 hour, and the final concentration of the antibody is preferably 0.15 mg / ml.

[0028] Collect peripheral blood mononuclear cells (PBMC) from the patient through a blood cell separator, transfer the collected blood sample to a centrifuge tube; 700g, centrifuge for 10 minutes, and absorb the upper layer of plasma for later use; use 0.9% normal saline blood sample to restore the sample to its original volume, Mix well; slowly add the diluted blood to Ficoll, 900g, and centrifuge for 20min; absorb the milky white mononuclear cell layer at the interface of the separation solution; centrifuge and wash twice and count; resuspend PBMC with serum-free medium, and adjust the cell concentration to 1.0 ×10 6 / ml.

[0029] After transferred to the cell cult...

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Abstract

The invention relates to a method used for in vitro proliferation of NK cells, and more specifically relates to a method used for realizing large-scaled proliferation of NK cells via autologous peripheral blood mononuclear cell in vitro cultivating. According to the method, proliferation and activation of NK cells are realized via different cytokine combination. The method comprises following steps: autologous peripheral blood mononuclear cells are inoculated in CD3 antibody and CD16 antibody pre-coated culture bottles, and are subjected to stimulation for 16 to 24h; and a serum-free medium containing IL-2 and IL-18 is added. According to the method, CD3 antibody and CD16 antibody are immobilized via coating, direct stimulating activation of NK cells is performed, so that toxicity of NK cells is increased greatly, activation and proliferation of NK cells are realized using two cytokines IL-2 and IL-18, proliferation multiple and cytotoxicity of NK cells are ensured, and culturing cost of NK cells is reduced greatly.

Description

[technical field] [0001] The invention relates to a method for using autologous peripheral blood mononuclear cells to culture and expand NK cells in large quantities in vitro, and in particular to a method for proliferating and activating NK cells by using different concentrations of cytokine combinations and treatment methods. [Background technique] [0002] Natural killer cells (NK) are cytotoxic lymphocytes belonging to the lymphocyte lineage that contain perforin and granzyme granules. Its recognition of target cells is not restricted by MHC, it can directly kill tumor cells without prior sensitization, and can also secrete cytokines to regulate the functions of other immune cells. It is the main bearer of the body's natural immunity and the core of acquired cellular immunity. Regulatory cells play an important role in tumor immunity, anti-viral infection and removal of non-self cells. Studies have proved that natural killer cells are not only an important component of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 叶永清柳运猛杨雪晶苏国新
Owner SHANGHAI CLAISON BIOTECH
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