Cell culture medium and method for generation of epithelial organoids from epithelial stem cells

Patent metadata

Field	Value
Publication number	US-12221630-B2
Application number	US-202017432001-A
Country	US
Kind code	B2
Filing date	Feb 18, 2020
Priority date	Feb 19, 2019
Publication date	Feb 11, 2025
Grant date	Feb 11, 2025

Abstract

The present invention provides a cell culture for obtaining an epithelial organoid, the cell culture comprising i) epithelial stem cells, or tissue fragments comprising said epithelial stem cells, ii) a basal medium for animal or human cells, iii) a Bone Morphogenetic Protein (BMP) inhibitor, iv) a mitogenic growth factor selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and keratinocyte growth factor (KGF), v) at least one soluble culture enhancer, wherein said at least one culture enhancer induces correct polarization of the cells in said cell culture within the developing organoid such as a laminin/entactin complex or entactin, and vi) a Wnt agonist if said epithelial stem cells, or tissue fragments comprising said epithelial stem cells are healthy cells, wherein said at least one soluble culture enhancer in said cell culture is a laminin/entactin complex in a concentration between 0.2 mg/ml and 3.4 mg/ml. A cell culture medium, an in-vitro method for obtaining an epithelial organoid, and an epithelial organoid obtained by said method are also disclosed.

First claim

The invention claimed is:

1. A cell culture for obtaining an epithelial organoid, the cell culture comprising:
i) epithelial stem cells, or tissue fragments comprising said epithelial stem cells,
ii) a basal medium for animal or human cells,
iii) a Bone Morphogenetic Protein (BMP) inhibitor,
iv) a mitogenic growth factor selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-alpha (TGFα), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and keratinocyte growth factor (KGF),
v) at least one soluble culture enhancer, wherein said at least one culture enhancer induces polarization of the cells in said cell culture that supports development of said epithelial organoid, and
vi) a Wnt agonist if said epithelial stem cells, or tissue fragments comprising said epithelial stem cells, are healthy cells,
wherein said at least one soluble culture enhancer in said cell culture is a complex comprising laminin and entactin at a combined concentration between 0.2 mg/mL and 1 mg/mL.

2. The cell culture according to claim 1 , wherein said cell culture does not contain a solidified 3D structure apart from said complex comprising laminin and entactin.

3. A cell culture medium for culturing epithelial stem cells, or tissue fragments comprising said epithelial stem cells, for obtaining an epithelial organoid, said cell culture medium comprising:
i) a basal medium for animal or human cells,
ii) a Bone Morphogenetic Protein (BMP) inhibitor,
iii) a mitogenic growth factor selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-alpha (TGFα), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and keratinocyte growth factor (KGF),
iv) at least one soluble culture enhancer, wherein said at least one culture enhancer induces polarization of the cells in said cell culture that supports development of said epithelial organoid, and
v) a Wnt agonist if said epithelial stem cells, or tissue fragments comprising said epithelial stem cells, are healthy cells,
wherein said at least one soluble culture enhancer in said cell culture medium is a complex comprising laminin and entactin at a combined concentration between 0.2 mg/mL and 1 mg/mL.

4. The cell culture medium according to claim 3 , wherein said cell culture does not contain a solidified 3D structure apart from said complex comprising laminin and entactin.

5. An in vitro method for obtaining an epithelial organoid, the method comprising culturing epithelial stem cells, or tissue fragments comprising said epithelial stem cells in the presence of
i) a basal medium for animal or human cells,
ii) a Bone Morphogenetic Protein (BMP) inhibitor,
iii) a mitogenic growth factor selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-alpha (TGFα), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and keratinocyte growth factor (KGF),
iv) at least one soluble culture enhancer, wherein said at least one culture enhancer induces polarization of the cells that supports development of said epithelial organoid, and
v) a Wnt agonist if said epithelial stem cells, or tissue fragments comprising said epithelial stem cells, are healthy cells,
wherein said at least one soluble culture enhancer in said cell culture is a complex comprising laminin and entactin at a combined concentration between 0.2 mg/mL and 1 mg/mL.

6. The in vitro method of claim 5 , wherein said at least one soluble culture enhancer is a complex of molecules comprising in parts by weight of 60-85% laminin, 5-30% collagen IV, 1-10% heparan sulfate proteoglycan, and 2-15% entactin at a total concentration between 0.2 mg/mL and 1 mg/mL.

7. The in vitro method according to claim 5 , wherein said soluble culture enhancer is initially present during the culturing at a concentration below solidification phase.

8. The in vitro method according to claim 5 , wherein said method does not comprise a step of providing a solidified three-dimensional (3-D) structure to said medium apart from said complex comprising laminin and entactin.

9. The in vitro method according to claim 5 , wherein the method comprises the step of passaging the cells in said cell culture medium, and wherein said passaging is performed by an automated process.

Assignees

• Miltenyi Biotec Bv & Co Kg

Inventors

• Agorku David Joel
• Hardt Olaf Thorsten
• Bosio Andreas
• Becker Kristin
• Eckardt Dominik

Classifications

• C12N2533/70

  Polysaccharides · CPC title

• C12N2533/54

  Collagen; Gelatin · CPC title

• C12N2533/52

  Fibronectin; Laminin · CPC title

• C12N2513/00

  3D culture · CPC title

• C12N2501/415

  Wnt; Frizzeled · CPC title