2d organoid for infection and culture of human diarrhea virus, and use of said 2d organoid

A technology for proliferation culture and organoids, which can be applied in the direction of virus, 3D culture, culture process, etc.

Pending Publication Date: 2019-04-19
KEIO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the cell culture system described in Non-Patent Document 1 cannot be used for research on human viruses because human viruses cannot be used, and the development of cell culture systems for human viruses is expected.

Method used

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  • 2d organoid for infection and culture of human diarrhea virus, and use of said 2d organoid
  • 2d organoid for infection and culture of human diarrhea virus, and use of said 2d organoid
  • 2d organoid for infection and culture of human diarrhea virus, and use of said 2d organoid

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Experimental program
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preparation example Construction

[0146] Examples of methods for producing ECM include methods using bound tissue cells, and the like. More specifically, after culturing ECM-producing cells such as fibroblasts, removing these cells and adding epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues containing them, ECM can be used as a base site.

[0147] Examples of ECM-producing cells include chondrocytes that mainly produce collagen and proteoglycans, fibroblasts that mainly produce type IV collagen, laminin, interstitial procollagen, and fibronectin, and that mainly produce collagen (type I, type III and type V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin and tenascin-C colonic myofibroblasts, etc. Alternatively, commercially available ECMs can also be used. Examples of commercially available ECMs include: extracellular matrix proteins (manufactured by Invitrogen), basement membrane preparations derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (for exampl...

experiment example 1

[0188] (Preparation of cell culture medium)

[0189] First, to commercially available Advanced DMEM / F-12 medium (manufactured by ThermoFicher SCIENTIFIC), human recombinant R-spondin 1 (manufactured by R&D systems) was added to a final concentration of 1 μg / mL, and noggin ( Peprotech) to a final concentration of 100 ng / mL, A83-01 (Tocris) was added to a final concentration of 500 nM (hereinafter referred to as "NRA medium").

[0190] Further, a culture medium was prepared in which the following combinations were added: Wnt3a at a final concentration of 300 ng / mL, IGF1 at a final concentration of 500 ng / mL (manufactured by Biolegend), FGF2 at a final concentration of 50 ng / mL (peprotech company), EGF (manufactured by Thermo Ficher SCIENTIFIC) at a final concentration of 50 ng / mL, SB202190 (manufactured by Sigma Aldrich) at a final concentration of 10 μM, and LY411575 (manufactured by Sigma Aldrich) at a final concentration of 1 μM.

[0191] ·WNRA+IGF1+FGF2 Medium

[0192] ·WE...

experiment example 2

[0203] (Proliferation of Norovirus Using Organoids)

[0204] "Cultivation of Intestinal Stem Cells"

[0205] Based on an ethical research plan approved by the Ethics Committee of the Faculty of Medicine, Keio University, normal mucosa was collected at least 5 cm away from the gastrointestinal tumor from healthy subjects and patients with gastrointestinal tumors who had obtained instructions and consent. Epithelial cells were extracted from the collected tissue by means of EDTA or Liberase TH, and embedded in Matrigel (registered trademark).

[0206] Matrigel (registered trademark) containing epithelial cells (hereinafter referred to as "intestinal stem cells") was seeded on a 48-well plate and cultured. Specifically, as follows.

[0207] The cultured intestinal stem cells were seeded on a 48-well plate together with 25 μL of Matrigel (registered trademark) (manufactured by BD Bioscience). 100 µL per well of the WNRA+IGF1+FGF2 medium prepared in (1) was added to each well, a...

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Abstract

A 2D organoid for infection and culture of human diarrhea virus epithelial cell stem cell, which is obtained by Step 1, in which: human intestinal epithelial stem cells, human intestinal epithelial cells, or a tissue that includes at least one of these cells is subjected to three-dimensional culturing in the extracellular matrix; and a 3D organoid is obtained, and Step 2, in which: the 3D organoids obtained in Step 1 are dispersed to prepare single cells; the single cells are subjected to monolayer culturing in the extracellular matrix; and a 2D organoid is obtained in which the epithelial cells, which include differentiated trophoblastic cells and caliciform cell, and which constitute part of the human intestinal cavity, have a single-layer structure.

Description

technical field [0001] The present invention relates to a 2D organoid for infection, proliferation and culture of human diarrheal disease virus and its use. More specifically, it relates to a 2D organoid for infection, proliferation and culture of human diarrhea virus, a 2D organoid culture for infection, proliferation and culture of human diarrhea virus, a method for producing human diarrhea virus, a human diarrhea virus SARS virus production kit, 2D organoid culture kit for infection, proliferation and culture of human diarrhea virus, and production method of 2D organoids for infection, proliferation and culture of human diarrhea virus. This application claims priority based on Japanese Patent Application No. 2016-163711 filed in Japan on August 24, 2016, the content of which is incorporated herein. Background technique [0002] Antiviral drugs that are highly effective in treating infections with human diarrheal viruses such as human norovirus do not yet exist. Most hum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00C12N15/09
CPCC12N7/00C12N2770/16051C12N5/0679C12N2501/415C12N2533/54C12N2533/90C12N2501/727C12N2503/00C12N2501/155C12N2500/80C12N5/068C12N2501/734C12N2501/15C12N2501/11C12N2501/105C12N2501/115C12N5/0018C12N5/0062C12N15/86
Inventor 佐藤俊朗片山和彦
Owner KEIO UNIV
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