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Method and kit for preparing dendritic cells

A technology of dendritic cells and cells, applied in the field of kit preparation, can solve the problems of ethics, the inability to apply transgenic technology, etc.

Inactive Publication Date: 2013-06-26
宁波高新区世纪开元生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, transgenic technology cannot be applied to clinical practice at present, and there are major ethical issues

Method used

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  • Method and kit for preparing dendritic cells
  • Method and kit for preparing dendritic cells
  • Method and kit for preparing dendritic cells

Examples

Experimental program
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Effect test

preparation example Construction

[0042] The medium used in the method for producing dendritic cells of the present invention is not particularly limited, and known media that can be used for culturing dendritic cells and the like can be used, for example, commercially available media can be appropriately selected and used. The above-mentioned medium may contain cytokines, appropriate proteins and other components in addition to its original constituents. Typically, the present invention uses media containing GM-CSF, IL-4 and LPS. The concentration of GM-CSF in the culture medium is 500-5000IU / mL, more preferably 1000-2000IU / mL. The concentration of IL-4 in the culture medium is 100-2000IU / mL, preferably 300-1000IU / mL. The concentration of LPS in the culture medium is 1-200ng / mL, preferably 10-100ng / mL.

[0043] Serum or plasma can be added to the medium for culturing. The amount of them added in the culture medium is not particularly limited, such as greater than 0% to 20% by volume, and the amount of seru...

Embodiment 1

[0062] In vitro culture of immature dendritic cells induced from peripheral blood mononuclear cells

[0063] 100mL peripheral blood from blood donors was centrifuged through Ficoll-Hypaque density gradient to obtain mononuclear cells. Peripheral blood mononuclear cells were resuspended in RPMI1640 medium and added to a 6-well plate to adhere to the wall. At 37°C, 5% CO 2 After incubation in the incubator for 90 min, unattached cells were washed and collected. Adherent cells were induced by adding complete medium RPMI1640, containing 5% autologous serum, 2000IU / mL GM-CSF, 1500IU / mL IL-4, 100ng / mL PMA, 1× non-essential amino acids, 1× sodium pyruvate, and placed for 37 °C, 5% CO 2 cultured in an incubator. On the third day of culture, the cells were replaced with fresh medium (containing 2000IU / mL GM-CSF, 1500IU / mL IL-4 and 100ng / mL PMA). The immature dendritic cells were harvested on the fifth day of culture.

[0064] Count the number of viable cells on the last day of cu...

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Abstract

The invention provides a method for preparing dendritic cells. The method comprises the process of culturing to obtain dendritic cells which high-express costimulatory molecules such as 4-1BBL, CD80, CD83 and CD86 in the presence of a human granulocyte-macrophage colony stimulating factor, interleukin-4, phorbol myristate acetate and a tumor necrosis factor alpha. The invention also provides a kit, a kit product obtained by adopting the method provided by the invention and an application of the kit product in preparation of the dendritic cells. The kit product can be applied to fundamental research and clinical application of the dendritic cells.

Description

technical field [0001] The invention relates to a preparation method of dendritic cells, preparation of a kit and the like. The invention relates to promoting the induction and proliferation of mononuclear cells in human peripheral blood, bone marrow and umbilical cord blood into dendritic cells, highly expressing co-stimulatory molecules, and activating body effector T lymphocytes, which can be used for various types of cancers including solid tumors and blood Immunotherapy in cancer. Background technique [0002] At present, cancer cell immunotherapy provides patients with the "fourth mode" following the three conventional treatments. It does not have the huge toxic and side effects caused by radiotherapy and chemotherapy, and achieves a relatively safe and dignified treatment that improves the quality of life of patients. Dendritic cells play a major role in this cellular immunotherapy and are used in clinical treatment. [0003] The 4-1BB ligand / 4-1BB signal transducti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784A61P35/00
Inventor 黄启明刘家森
Owner 宁波高新区世纪开元生物技术有限公司
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