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Method for evaluating Anti-infective drugs, vaccines, etc. using immortalized monocytic cells and induced cells

A mononuclear cell differentiation induction technology, applied in biochemical equipment and methods, botany equipment and methods, microorganisms, etc., can solve the problem of inability to obtain attenuated mutant strains, and achieve the effect of small error

Pending Publication Date: 2021-04-02
迈凯恩技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this way, it is known that it is usually impossible to obtain attenuated mutant strains without repeated cultivation

Method used

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  • Method for evaluating Anti-infective drugs, vaccines, etc. using immortalized monocytic cells and induced cells
  • Method for evaluating Anti-infective drugs, vaccines, etc. using immortalized monocytic cells and induced cells
  • Method for evaluating Anti-infective drugs, vaccines, etc. using immortalized monocytic cells and induced cells

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0089]

[0090]In the present invention, a monocyte which becomes a raw material can be obtained from a method such as a fibroblast or the like from a plurality of cellular cell differentiated induced methods, from a plurality of stem cell differentiated induced methods. When a monocyte is extracted from a tip blood, it is known that a method of preparing a cell expressing CD14 molecule in human tipmed blood is known. For example, the use of normal saline, phosphate buffer, salt water or Hanks buffer, etc., using a physiological saline, phosphate buffer, and Hanks buffer. The diluted blood was carefully laminated on the FICOLL (Registered Trademark) (GE Healthcare) (GE Healthcare) (GE Healthcare), centrifugation at 15 to 30 ° C, 500 to 1000 × g. The yellow plasma with the transparent FICOLL intermediate white strip layer was recovered by a lymphocyte and monocytes. The recovered tip blood mononuclear cell component can also be used further after further cleaning. Cell cells can be obta...

Embodiment 1

[0137](Example 1) Human Tip Source Permanent Monuclear Cell (CD14-ML) production and dendritic cell production (human tipped blood source immortal mononuclear cell production)

[0138]Human tipped sources of immortal mononuclear cells have been made (WO2012 / 043651 and Japanese special opening 2017-131136). In detail, the CD14 positive component is taken out from the end of the human tip and introduced as a gene that is reported as a mouse endogenous hematopoietic stem cell (Myc, Myb, HOB8, TLX1, E2A-PBX1, MLL, LJX2, RARA, HOXA9, NOT1, V -RAF / V-MYC, MyST3-NCoA2, EVI1, HOXB6, HOSB4, β-catenin, ID1), or genes in the forever-mononuclear cell report. The immortalized monocytes are cultured in α-MEM medium containing 20% ​​FBS, 50 ng / ml m-CSF and 50 ng / ml GM-CSF, from starting to cultivate a proliferative cell, using commercially available Cell preservation fluid is stored in liquid nitrogen.

[0139](Confirmation of the obtained cells)

[0140]To confirm that the prepared cells are monocy...

Embodiment 2

[0145](Example 2) Made of immortal mononuclear cells (IPS-ML) from human induced multi-capable cells

[0146]Human-induced monocytes (IPS) sources of human immortal mononuclear cells have been made (WO2012 / 043651 and Japanese special opening 2017-131136). In detail, undifferentiated IPS cells were inoculated with substrate equipases such as laminin 511 (Laminin-511) as a base film, and the α-MEM medium (containing 20% ​​FBS) was used. Differentiation induction culture. Then, a 1-day α-MEM medium (containing 20% ​​FBS) continued to cultivate every 3 days. After 18 days of starting differentiation, Trypsin-Edta (ethylenediamine tetracetate)-collagenase (37 ° C, 60 minutes) cells, lysis, recovery, by pipetting (PIPETTING) Operation and production of cell suspensions. Next, the cells from a culture dish from one diameter of 10 cm were suspended in 10 ml of DMEM (containing 10% FBS) medium, and inoculated in two diameters without feeding cells and did not have gelatincoat. 10 cm of petriper...

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Abstract

The present invention was made in view of problems relating to stability, reproducibility, economics, and ease of operation in monocyte- or dendritic cell-mediated infectious microorganism research and provides a method for the maintenance culture of monocyte- or dendritic cell-mediated infectious microorganisms using monocytes having proliferative capacity. The present invention is based on the discovery that the dengue virus can infect and proliferate efficiently in human monocytic cells capable of proliferation obtained by introducing a gene into CD14-positive cells and cells (for example,dendritic cells) having phagocytic capacity obtained by inducing differentiation of the monocytic cells. Thereby provided is a novel method for evaluating pharmaceuticals such as compounds and vaccines for the treatment of monocyte- or dendritic cell-mediated infectious microbial infections.

Description

Technical field[0001]The present invention relates to a monocyte or dendritic cell-mediated infectious microorganism, or an evaluation method of the treatment of drugs in the microbial infectious disease.Background technique[0002]In the infectious disease study, the target pathogens were studied using a constructed cell line when proliferated by monocytes or dendritic cells. For example, the dengue fever virus is a virus that has been infected for long-term research. According to the experience of the K562 cell line of human chronic myeloid leukemia, the Nero cell line of the kidney epithelial cells of African macaque, the Syrian hamster kidney source BHK Cell lines such as cell lines were studied. However, although the evaluation of these cell lines has an advantage that it is easy to perform, there is no problem that does not necessarily reflect infections and proliferation in the human body.[0003]The use of human stems monocytes and dendritic cells can be analyzed more than organ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0784C12N5/0786C12N7/00C12N7/04C12N15/12C12Q1/04G01N33/15G01N33/50A61K39/12A61P31/12
CPCC12N7/00C12Q1/04C12N2770/24131G01N33/5008G01N33/5047G01N33/5073C12N2770/24121Y02A50/30A61K39/12A61K2039/5252C12N5/10C12N2770/24134C12N2770/24162G01N2496/00
Inventor 宫﨑和雄山中敦史冈田稔千住觉
Owner 迈凯恩技术有限公司
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