Type 1 polarized dendritic cells and inducing method and application thereof

A dendritic cell and cell technology, applied in the biological field, can solve the problems of a large number of cytokines and inducing reagents, quality control workload, high cost, etc., and achieve the effects of being beneficial to quality control, reducing production costs, and reducing use.

Inactive Publication Date: 2016-01-06
SHANGHAI LONGYAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The best solution in the prior art can obtain DC1 cells with high expression of IL12, but both require a large amount of cytokines and induction reagents, which are expensive
In addition, there may be differences in potency between cytokine batches, which brings a lot of burden to the quality control work of the preparation

Method used

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  • Type 1 polarized dendritic cells and inducing method and application thereof
  • Type 1 polarized dendritic cells and inducing method and application thereof
  • Type 1 polarized dendritic cells and inducing method and application thereof

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preparation example Construction

[0032] Then, the preparation of DC1 cells is carried out: monocytes in peripheral blood are separated, after the cells are attached to the wall for a short time, the suspension cells are removed, and the inducers GM-CSF and IL-4 are cultured for five days to obtain mature DC cells. Mature DC cells were further induced with a medium containing INFγ and NK cell supernatant for two days to promote the differentiation of DC cells into DC1 cells. Compared with the mature DC cells prepared by the conventional method, the DC1 cells prepared by the method of the present invention are positive for multiple surface markers of CCR7, CD70, HLA-DR, CD83, CD86 and other markers related to immune activation (such as figure 1 ).

[0033] The above processes are all carried out in vitro, thereby obtaining a cell population enriched with DC1 cells. After CD40L stimulation, DC1 cells highly express IL12. IL12 plays a vital role in the specific immune activation process of T cells. The DC1 induced ...

Embodiment 1

[0039] Preparation of natural killer cell culture supernatant (NaturalKillercell, NK)

[0040] Isolation of peripheral blood mononuclear cells refers to the method described by Jonuleit et al. (GeneTher. 200310(3)). First, peripheral blood mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation.

[0041] According to density 3×10 6 / ml transfer to a new culture flask, add IL-2 (2000U / ml) and IL15 (2ng / ml). Add the medium every two to three days and add the same amount of factors. Collected on the twelfth day. After the induction is completed, NK cells press 1-3×10 6 / ml inoculated into the medium RPMI1640, add IFNa (1000UI / ml) and add 0.5-1.5×10 5 Inoculate K562 cells (can be replaced by K562-NK cells) mixed culture at a density of / ml, collect the medium supernatant during activation for 24-48 hours, filter and freeze at -80 degrees or directly use for the preparation of DC1 cells.

Embodiment 2

[0043] Preparation of Type I Polarized Dendritic Cells from Adult Peripheral Blood

[0044] Isolation of peripheral blood mononuclear cells refers to the method described by Jonuleit et al. (GeneTher. 200310(3)). First, peripheral blood mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation, and plasma was collected for subsequent culture.

[0045] According to density 3.0×10 6 / ml, inoculate the cells into a T75 culture flask, and add 10% plasma to the expansion medium containing 1000U / ml IL-2, and place it at 37.0℃, saturated humidity, and 5% CO 2 Cultivate in the environment. After culturing for 3 hours, the suspended lymphocytes were washed away, and a medium containing GM-CSF and IL-41000 U / ml and 10% plasma was added. Adherent cells were cultured on the 3rd day and half of the medium was replaced on the 5th day.

[0046] On the sixth day of culture, change the medium containing 5% plasma and add 10% of NK cell supernatant, and add IFNγ at a final c...

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Abstract

The invention provides type 1 polarized dendritic cells and an inducing method and application thereof. A novel DC1 preparation method is built through the assistant function of Natural Killer cells of umbilical cord blood in the adaptive immunity activating process of DCs, and in the preparation process, usage of cell factors is reduced, the cost is lowered, and quality control is improved. The invention further provides a method for applying the prepared DC1s to preparation of CTLs with the tumor antigen specificity; after the DC1s are loaded with tumor specific antigens, part of the cells are transfused to a patient body, the other part of the cells are used for in-vitro inductive cytotoxic T lymphocytes (CTL) with the tumor antigen specificity, and the immunoreaction of the tumor specificity is activated through the mode of combining active induction with passive induction in vivo and vitro.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and mainly relates to a method for culturing type I polarized dendritic cells and related applications. Background technique [0002] DC cells are the only full-time antigen presenting cells in the immune system of mammals. Its main function is to ingest, process and present antigens, and activate or regulate adaptive immune responses. Mature DC cells highly express MHC-I and MHC-II molecules. MHC molecules bind to tumor antigens captured and processed by cells to form peptide-MHC molecular complexes, which are presented to T cells to activate MHC-I Class-restricted CD8-positive cytotoxic T cells (Cytotoxic Tlymphocyte, CTL) and MHC-Ⅱ-restricted CD4-positive type 1 T helper cells (type1Thelper, Th1). Mature DC cells highly express CD80 / B7-1, CD86 / B7-2, CD40 and other costimulatory molecules, which provide T cells with a second signal necessary for their activation. Mature DC cells also have a l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784A61K35/15A61P35/00
Inventor 李伟叶圣勤汪鑫瞿苏
Owner SHANGHAI LONGYAO BIOTECH CO LTD
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