Cell culture medium for culturing organoid, culture method, and organoid

A technology of organ culture and culture method, applied in the direction of cell culture active agent, chemical instrument and method, culture process, etc., can solve the problem of inability to realize long-term culture of intestinal epithelial cells, etc.

Active Publication Date: 2019-03-01
KEIO UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In addition, long-term culture of intestinal epithelial cells has long been impossible

Method used

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  • Cell culture medium for culturing organoid, culture method, and organoid
  • Cell culture medium for culturing organoid, culture method, and organoid
  • Cell culture medium for culturing organoid, culture method, and organoid

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preparation example Construction

[0157] As a method for producing ECM, for example, a method using connective tissue cells and the like may be mentioned. More specifically, ECM can be used as a base by culturing ECM-producing cells such as fibroblasts, removing these cells, and adding epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues containing these cells.

[0158] 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 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 be used. As a commercially available ECM, for example, extracellular matrix protein (manufactured by Invitrogen), basement membrane preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (such as Matrigel (...

Embodiment 1

[0202] (1) Preparation of cell culture medium for organoid culture

[0203] First, human recombinant R-spondin 1 (manufactured by R&D systems) was added to a commercially available modified DMEM / F-12 medium (manufactured by Thermo Ficher SCIENTIFIC) at a final concentration of 1 μg / mL at a final concentration of 100 ng. Noggin (manufactured by Peprotech) was added conditionally at a concentration / mL, and A83-01 (manufactured by Tocris) was added conditionally at a final concentration of 500 nM. Then, a medium (hereinafter also referred to as "WNRA medium") was prepared in which the W-Wnt3a / HEK-derived culture supernatant cultured in a serum-containing medium was added under the condition that the final concentration of Wnt3a was 300 ng / mL.

[0204] Then, at least one of epidermal growth factor (Epiregulin) (manufactured by Biolegend) at a final concentration of 500 ng / mL, IGF1 (manufactured by Biolegend) at a final concentration of 500 ng / mL, or FGF2 (manufactured by Peprotech...

Embodiment 2

[0215] (1) Preparation of cell culture medium for organoid culture

[0216] First, EGF (manufactured by Thermo Ficher SCIENTIFIC) was added at a final concentration of 50 ng / mL to a commercially available modified DMEM / F-12 medium (manufactured by Thermo Ficher SCIENTIFIC) and added at a final concentration of 100 ng / mL Noggin (manufactured by Peprotech) was supplemented with A83-01 (manufactured by Tocris) at a final concentration of 500 nM (hereinafter also referred to as "ENA medium").

[0217] (2) Culture of epithelial tumor cells derived from colorectal tumors

[0218] Epithelial tumor cells were obtained by the same method as in (2) of Example 1. Next, the epithelial tumor cells were inoculated into a 48-well plate together with 25 μL of Matrigel (registered trademark) (manufactured by BD Biotech). 250 μL of the ENA medium prepared in (1) was added to the wells inoculated with epithelial tumor cells, and cultured at 37° C. with an oxygen concentration of 1% (hereinafte...

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Abstract

A cell culture medium for culturing an organoid which comprises at least two members selected from the group consisting of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and epiregulin (EREG), and at least one member selected from components i) to iii): i) a Wnt agonist; ii) a bone morphogenesis protein (BMP) inhibitor; and iii) a transforming growth factor-beta (TGF-beta)inhibitor.

Description

technical field [0001] The invention relates to a cell culture medium for organoid culture, a culture method and an organoid. [0002] This application claims priority based on Japanese Patent Application No. 2016-099995 for which it applied to Japan on May 18, 2016, and uses the content for this application. Background technique [0003] The intestinal tract is the organ with the largest area in contact with the outside world in the human body, and it has functions such as digestion and absorption that are essential for maintaining life. Much of the gut's function is taken care of by the gut epithelium that covers its lining. The intestinal epithelium is composed of two regions: villi formed by three lineages of differentiated cells (mucous cells, absorptive epithelial cells, and endocrine cells), and crypts formed mainly by undifferentiated proliferating cells. In the intestinal glands of the small intestine, there are Paneth cells that produce antimicrobial peptides at ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/00C12N5/071C12N5/074C12N5/09C07K14/475C07K14/51
CPCC12N2501/105C12N2501/115C12N2501/415C12N2501/155C12N2501/15G01N2500/10C12N5/0697G01N33/5011G01N33/5088C12N2502/30C12N2502/03C12N2502/09C12N2500/02C12N2501/727C12N2503/04C07K14/51C07K14/475C12N1/00C12N5/0693C12N2500/90C12N2501/998
Inventor 佐藤俊朗股野麻未
Owner KEIO UNIV
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