Preparation method of high-activity antigen-loaded dendritic cell

A dendritic cell, high activity technology, applied in the biological field, can solve the problems of low antigen presentation ability, insufficient killing activity of CTL cells, affecting the effect of tumor treatment, etc., and achieve the effect of improving the killing activity

Active Publication Date: 2013-04-03
HANGZHOU CONVERD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is that due to the weak antigenicity of tumor antigens in the dendritic cells prepared in the prior art, the ability of DC to present antigens to them is also low, so that the killing activity of the CTL cells produced by it is insufficient. , and then affect the technical problems of the therapeutic effect on tumors, and then provide a preparation method of highly active antigen-loaded dendritic cells, which can not only ensure the number of dendritic cells, but also improve the ability of dendritic cells to load tumor antigens quantity

Method used

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  • Preparation method of high-activity antigen-loaded dendritic cell

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Embodiment 1

[0020] Collect and isolate mononuclear cells from peripheral blood, add DC serum-free culture medium, and then add cytokines IL-4 and GM-CSF to the culture medium, adjust the concentration of IL-4 to 5ng / ml, and the concentration of GM-CSF to 10ng / ml, then cultured in a 37°C, 5% CO2 incubator, collected dendritic cells 6 days later; then added the malignant melanoma cell line lysate and Mycobacterium tuberculosis pure protein derivative (PPD) as antigens , adjust the PPD concentration to 0.1u / ml, and continue culturing for 2 days to obtain highly active antigen-loaded dendritic cells.

Embodiment 2

[0022] Collect and isolate mononuclear cells from peripheral blood, add DC serum-free culture medium, and then add cytokines IL-4 and GM-CSF to the culture medium, adjust the concentration of IL-4 to 100ng / ml, and the concentration of GM-CSF to 500ng / ml, then cultured in a 37°C, 5% CO2 incubator, and collected dendritic cells 6 days later; then added the lysate of malignant melanoma cell lines and the pure protein derivative of Mycobacterium tuberculosis (PPD) as antigens , adjust the PPD concentration to 2u / ml, and continue to culture for 2 days to obtain highly active antigen-loaded dendritic cells.

Embodiment 3

[0024] Collect and isolate mononuclear cells from peripheral blood, add DC serum-free culture medium, then add cytokines IL-4 and GM-CSF to the culture medium, adjust the concentration of IL-4 to 50ng / ml, and the concentration of GM-CSF to 255ng / ml, then cultured in a 37°C, 5% CO2 incubator, collected dendritic cells 6 days later; then added as antigen malignant melanoma cell line lysate and Mycobacterium tuberculosis pure protein derivative (PPD) , adjust the PPD concentration to 0.5u / ml, and continue to culture for 2 days to obtain highly active antigen-loaded dendritic cells.

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Abstract

The invention discloses a preparation method of a high-activity antigen-loaded dendritic cell. The method comprises the following steps of a, collecting and separating single karyocyte from peripheral blood; b, adding the single karyocyte obtained in the step a into DC (dendritic cell) serum-free medium, adding cytokines IL-4 and GM-CSF into the medium, putting in a 5 percent CO2 culture tank at 37 DEG C to culture, and collecting the dendritic cell after 6 days; and c, adding corresponding antigen and mycobacterium tuberculosis purified protein derivative (PPD) into the medium containing the dendritic cell collected in the step b, and keeping on culturing to obtain the high-activity antigen-loaded dendritic cell. According to the preparation method, PPD is added when tumor antigen is loaded to have an effect similar to immunologic adjuvant, so that the capability of DC in treating tumor antigen can be improved, and the killing activity of CTL (cytotoxic lymphocyte) generated by stimulation of the cells to tumor cells can be indirectly improved.

Description

technical field [0001] The invention relates to a preparation method of highly active antigen-loaded dendritic cells, belonging to the field of biotechnology. Background technique [0002] Tumor biological therapy is the fourth treatment mode after surgery, radiotherapy and chemotherapy. Tumor cell immunotherapy is one of the most effective methods in tumor biotherapy. Due to the polymorphism of tumor cells, it is impossible to rely on only one method to cure tumors. Antigen-presenting cells (antigen-presenting cells, APCs) are a type of immune cells that can ingest, process, and present antigens to lymphocytes, and are an indispensable part of the production and preparation of specific tumor killer cells , which plays an irreplaceable role in tumor immunotherapy. Dendritic cells (DC) are one of the antigen-presenting cells present in peripheral blood, skin, lymphoid organs, and thymus. They are enriched in lymphoid and non-lymphoid tissues and have strong antigen-presenti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784
Inventor 高岱清于华马伟赵鹏解西河魏晓芳李长优孙伟红
Owner HANGZHOU CONVERD CO LTD
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