Method for production of dendritic cell

一种树突状细胞、细胞的技术，应用在生物化学设备和方法、动物细胞、脊椎动物细胞等方向，能够解决无法达到理想治疗效果、不能获取DC、DC前体细胞数量有限等问题

Inactive Publication Date: 2010-06-23
DNAVEC CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical trials also believe that the amount of inoculated DC greatly affects the therapeutic effect, but due to the patient's condition, the number of DC progenitors that can be extracted is mostly extremely limited, and a sufficient number of DC cannot be obtained, so the ideal amount cannot be achieved. The therapeutic effect of

Method used

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  • Method for production of dendritic cell
  • Method for production of dendritic cell
  • Method for production of dendritic cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0206] The present invention will be further described below through examples, but the present invention is not limited to these examples. In addition, all the documents cited in this specification are incorporated as a part of this specification.

[0207] The composition components of the FS36 administration group, the GMSCF administration group and the GMIL-4 administration group in Examples 1, 2, 4, 5 and the figures of the examples are as follows.

[0208] FS36 administration group: containing Flt-3 ligand (20ng / ml), stem cell factor (Stem cell factor; SCF) (10ng / ml), IL-3 (10ng / ml), IL-6 (10ng / ml) ( Abbreviated as FS36), RPMI1640 with 10% FBS.

[0209] GMIL-4 administration group: RPMI1640 containing GM-CSF (20 ng / ml), IL-4 (20 ng / ml), and 10% FBS.

[0210] GMSCF administration group: RPMI1640 containing GM-CSF (20 ng / ml), SCF (10 ng / ml), and 10% FBS.

[0211] The GMIL-4 administration group (1), the GMIL-4 administration group (2), the GMSCF administration group, the ...

Embodiment 6

[0217] (1) iDC treatment, (2) SeV / dF treatment, and (3) LPS treatment in Example 6 and the drawing of the example are as follows.

[0218] (1) iDC treatment: Incubate for 2 days in the medium at the following concentrations.

[0219] IMDM with 10% FBS

[0220] (2) SeV / dF treatment: Incubate for 2 days in the medium at the following concentrations.

[0221] IMDM containing F gene deletion Sendai virus (moi=50), 10% FBS

[0222] (3) LPS treatment: Incubate for 2 days in the medium at the following concentration.

[0223] IMDM containing LPS (1 μg / ml), 10% FBS

[0224] This experiment uses the following LPS.

[0225] SIGMA Catalog No.L7895-1MG (biological source = Salmonella typhosa)

[0226] (4) Poly(I:C) treatment: Incubate for 2 days in the medium at the following concentration.

[0227] IMDM containing Poly(I:C) (100 μg / ml), 10% FBS

[0228] (5) CpG treatment: Incubate for 2 days in the medium at the following concentration.

[0229] IMDM containing CpG (10 μg / ml), 10...

Embodiment 1

[0234] [Example 1] Confirmation of Dendritic Cell (DC) Progenitor Expansion and Differentiation of Cytokines

[0235] First, hematopoietic precursor cells (SpinSep mouse hematopoietic progenitor enrichment kit, StemCelltechnologies, Canada) were extracted from mouse (C3H) femur and tibia bone marrow by negative selection. The precursor cells were divided into three groups including FS36 administration group, GMIL-4 administration group and GMSCF administration group for culture. cultured by 2.4 x 10 5 From the beginning, the cells were subcultured every 3 to 4 days at a concentration of less than 2 million cells / ml for 6 weeks. Modulation of dendritic cell (DC) precursor cells during culture ( figure 1 ), count the number of cells, confirm the rate of expansion, and perform FACS analysis after staining with anti-CD11b-FITC, anti-CD11c-PE, anti-c-kit-PE, and anti-CD131-PE, and confirm the differentiation ability of the above-mentioned precursor cells ( image 3 ).

[0236] ...

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Abstract

The invention provides a method for producing a dendritic cell (DC), which involves the step of culturing a DC precursor cell in the presence of two or more cytokines; a DC produced by the method; and use of the DC. The method can produce a large quantity of a DC precursor cell which is highly capable of being differentiated into a DC. The method can also produce a large quantity of a DC from a small quantity of a DC precursor cell. Therefore, it becomes possible to readily increase the number of DCs to be administered in the anti-tumor immunotherapy or the treatment of an infectious disease utilizing a DC, resulting in the enhancement of the efficacy of a DC vaccine.

Description

technical field [0001] The invention relates to a preparation method of dendritic cells, and provides the prepared dendritic cells and a utilization method thereof. Background technique [0002] Dendritic cell (DC) is one of the antigen-presenting cells (APC) present in peripheral blood, skin, lymphoid organs and thymus, and is enriched in lymphoid tissue and non-lymphoid tissue (refer to Steinman, R.M.1991 , Ann. Rev. Immunol. 9:271; Banchereau, J.B. and R.M. Steinman, 1998, Nature 392:245). Dendritic cells have a strong ability to present antigens, which can express antigenic peptides to class I and class II dendritic cells, and activate CD4 and CD8 T cells, respectively, thereby inducing in vivo response to specific antigens (pathogenic microbial antigens) , tumor antigen, transplant antigen, etc.) immune response. [0003] The powerful immune-inducing function of DC is very suitable for immunotherapy (DC treatment) of various tumors. Previous reports by the present in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/0784
CPCC12N2501/125C12N2501/23C12N5/0639C12N2501/22C12N2501/26A61K39/4615A61K2239/31A61K39/464499A61K39/4622C12N5/00
Inventor 井上诚长谷川护米满吉和原田结
Owner DNAVEC CORP
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