Liver organoid, uses thereof and culture method for obtaining them

Patent metadata

Field	Value
Publication number	US-9765301-B2
Application number	US-201113812614-A
Country	US
Kind code	B2
Filing date	Jul 29, 2011
Priority date	Jul 29, 2010
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

Abstract

The invention relates to a liver organoid, uses thereof and method for obtaining them.

First claim

The invention claimed is:

1. A method for obtaining a liver organoid comprising Lgr5+ epithelial stem cells, wherein said method comprises:
culturing and expanding Lgr5+ epithelial stem cells from a liver fragment or a liver biliary duct in contact with an extracellular matrix in the presence of a medium, the medium comprising a basal medium for animal or human cells to which is added: a Wnt agonist, EGF, an FGF and Nicotinamide in amounts sufficient to form a liver organoid comprising Lgr5+ epithelial stem cells which are capable of at least 5 passages in vitro.

2. The method according to claim 1 , wherein after at least one day, the medium is changed to a second medium.

3. The method according to claim 2 , wherein the second medium is a second expansion medium comprising a basal medium to which is added: EGF, an FGF, an HGF, Nicotinamide and an Rspondin.

4. The method according to claim 2 , wherein the second medium is a differentiation medium comprising a basal medium for animal or human cells to which is added EGF, FGF and/or HGF, and a Notch inhibitor.

5. The method according to claim 2 , wherein the method comprises culturing said epithelial stem cells in a cell culture medium comprising or consisting of a basal medium for animal or human cells to which is added EGF, a BMP inhibitor, R-spondin and Wnt; and subsequently in a second expansion medium which comprises or consists of a basal medium for animal or human cells to which is added: EGF, an FGF, and HGF, Nicotinamide, and an R-spondin; and subsequently in a differentiation medium which comprises or consists of a basal medium for animal or human cells to which is added: EGF, FGF and/or HGF, a TGF-beta inhibitor, and a Notch inhibitor.

6. The method according to claim 1 , wherein the Wnt agonist is selected from the group consisting of: a Wnt family member, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, Norrin and a GSK-inhibitor.

7. The method according to claim 1 , wherein one or more of a BMP inhibitor, a TGF-beta inhibitor and an HGF are added to the medium.

8. The method according to claim 7 , wherein a TGF-beta inhibitor is added to the medium and wherein the TGF-beta inhibitor is a small molecule inhibitor selected from the group consisting of: A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208 and SJN 2511.

9. The method according to claim 1 , wherein the culture medium further comprises HGF.

10. A method for obtaining a liver organoid comprising Lgr5+ epithelial stem cells, wherein said method comprises:
culturing and expanding Lgr5+ epithelial stem cells from a liver fragment or a liver biliary duct in contact with an extracellular matrix in the presence of a medium, the medium comprising a basal medium for animal or human cells to which is added: a Wnt agonist, EGF, an HGF and Nicotinamide in amounts sufficient to form a liver organoid comprising Lgr5+ epithelial stem cells, which are capable of at least 5 passages in vitro.

11. The method according to claim 10 , wherein the culture medium further comprises FGF.

Assignees

• Huch Ortega Meritxell
• Clevers Johannes Carolus
• Koninklijke Nederlandse Akademie Van Wetenschappen

Inventors

• Huch Ortega Meritxell
• Clevers Johannes Carolus

Classifications

• A61P1/16

  for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics · CPC title

• C12N2501/415

  Wnt; Frizzeled · CPC title

• C12N2501/119

  Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10 · CPC title

• A61K35/407 Primary

  Liver; Hepatocytes · CPC title

• C12N2500/38

  Vitamins · CPC title