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Cell culture medium, culture method, and organoid

A culture method and culture medium technology, which is applied in the direction of cell culture medium, cell culture active agent, cell culture support/coating, etc., and can solve problems such as the inability to achieve long-term culture of intestinal epithelial cells

Active Publication Date: 2018-12-21
KEIO UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In addition, long-term culture of intestinal epithelial cells has not been possible for a long time

Method used

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

Examples

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preparation example Construction

[0059] As a method for producing a Wnt protein, for example, a method of producing it using a Wnt protein-expressing cell is mentioned. In Wnt protein-expressing cells, the source of the cells (biological species, culture form, etc.) is not particularly limited, as long as it is a cell that stably expresses Wnt protein, and may be a cell that transiently expresses Wnt protein. Examples of Wnt protein-expressing cells include L cells stably expressing mouse Wnt3a (ATCC CRL-2647), L cells stably expressing mouse Wnt5a (ATCC CRL-2814), and the like. In addition, Wnt protein-expressing cells can be prepared using known gene recombination techniques. That is, Wnt protein-expressing cells can be prepared by inserting DNA encoding a desired Wnt protein into a known expression vector and introducing the resulting expression vector into an appropriate host cell. The nucleotide sequence of a gene encoding a desired Wnt protein can be obtained from known databases such as GenBank.

[0...

Embodiment 1

[0161] (1) Preparation of Wnt3a-Afamin complex

[0162] Utilizing the fact that fetal calf serum contains bovine Afamin, cells transfected with only Wnt3a are cultured in serum-containing medium, and secreted Wnt3a and Afamin automatically form a stable complex, thereby preparing a Wnt3a-Afamin complex. That is, cells expressing Wnt3a with a tag sequence at the N-terminus (W-Wnt3a / HEK) were cultured in a medium containing 10% fetal calf serum for 5 to 7 days using a petri dish or a multi-layer flask, and recovered and cultured. clear. Next, the recovered culture supernatant was centrifuged and passed through a filter (0.22 μm). Take 220 mL from the collected culture supernatant. Next, 3 mL of P20.1 antibody agarose was added to 200 mL of the recovered culture supernatant, and after rotating and mixing at 4° C. for 3 hours, the medium was passed through the empty column to collect the agarose. It should be noted that the P20.1 antibody is an antibody that specifically re...

Embodiment 2

[0171] (1) Preparation of cell culture medium

[0172] To commercially available Advanced DMEM / F-12 medium (manufactured by Thermo Ficher SCIENTIFIC), EGF (manufactured by Thermo Ficher SCIENTIFIC) was added so that the final concentration became 50 ng / mL so that the final concentration became 100 ng / mL. Noggin (manufactured by Peprotech) was added in such a manner that A83-01 (manufactured by Tocris) (hereinafter referred to as "ENA medium") was added so that the final concentration became 500 nM.

[0173] In addition, EGF (manufactured by Thermo Ficher SCIENTIFIC) was added to a commercially available Advanced DMEM / F-12 medium (manufactured by Thermo Ficher SCIENTIFIC) so that the final concentration became 50 ng / mL so that the final concentration became 100 ng / mL. Noggin (manufactured by Peprotech) was added in mL, A83-01 (manufactured by Tocris) was added so that the final concentration became 500 nM, and SB202190 (manufactured by Sigma Aldrich) (hereinafter referred...

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Abstract

The present invention provides a cell culture medium in which an epithelial stem cell, a cell carcinoma cell or a tissue containing at least one of the aforementioned cells can be cultured in the absence of serum for a long period. The cell culture medium according to the present invention contains: a Wnt agonist composed of a complex of Wnt protein and afamin, which is a substance capable of stabilizing the Wnt protein, and R-spondin; and at least one component selected from the group consisting of a division promoting growth factor, a bone morphogenetic factor (bone morphogenetic protein; BMP) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor and a p38 inhibitor.

Description

technical field [0001] The invention relates to a cell culture medium, a culture method and an organoid. [0002] This application claims priority based on Japanese Patent Application No. 2016-029060 filed in Japan on February 18, 2016, and uses the content here. Background technique [0003] The intestinal tract is the organ with the largest contact area with the outside world in the human body, and it has functions such as digestion and absorption that are indispensable for maintaining life. Much of the intestinal function is undertaken by the intestinal epithelium that covers its lining. The intestinal epithelium is composed of two compartments: villi, which contain differentiated cells of three systems (mucus-producing cells, absorptive epithelial cells, and endocrine cells), and crypts, which mainly contain undifferentiated proliferating cells. In small intestinal crypts, antimicrobial peptide-producing Paneth cells are present at the bottom of the crypts. Recently, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C12N5/0775C12N5/09C12N5/10
CPCC12N5/00C12N5/10C12N5/0037C12N5/0677C12N5/0695C12N2501/11C12N2501/117C12N2501/155C12N2501/415C12N2533/90A61P1/00
Inventor 佐藤俊朗股野麻未杉本真也髙木淳一
Owner KEIO UNIV
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