Liver organoid, uses thereof and culture method for obtaining them

• US 9,765,301 B2
• Filed: 07/29/2011
• Issued: 09/19/2017
• Est. Priority Date: 07/29/2010
• Status: Active Grant

• Current Assignee: Koninklijke Nederlandse Akademie Van Wetenschappen
• Original Assignee: Koninklijke Nederlandse Akademie Van Wetenschappen
• Inventors: Huch Ortega, Meritxell, Clevers, Johannes Carolus
• Primary Examiner(s): Underdahl, Thane
• Assistant Examiner(s): Pyla, Yvonne
• Application Number:
  US13/812,614
• Publication Number:
  US 20130189327A1
• Time in Patent Office: 2,244 Days
• Field of Search: None
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells

Abstract

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

Claims

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.