US 12,221,630 B2
Cell culture medium and method for generation of epithelial organoids from epithelial stem cells
David Joel Agorku, Bergisch Gladbach (DE); Olaf Thorsten Hardt, Bergisch Gladbach (DE); Andreas Bosio, Cologne (DE); Kristin Becker, Bergisch Gladbach (DE); and Dominik Eckardt, Bergisch Gladback (DE)
Assigned to MILTENYI BIOTEC B.V. & CO. KG, Bergisch Gladbach (DE)
Appl. No. 17/432,001
Filed by MILTENYI BIOTEC B.V. & CO. KG, Bergisch Gladbach (DE)
PCT Filed Feb. 18, 2020, PCT No. PCT/EP2020/054150
§ 371(c)(1), (2) Date Aug. 18, 2021,
PCT Pub. No. WO2020/169551, PCT Pub. Date Aug. 27, 2020.
Claims priority of application No. 19157908 (EP), filed on Feb. 19, 2019.
Prior Publication US 2022/0135943 A1, May 5, 2022
Int. Cl. C12N 5/071 (2010.01)
CPC C12N 5/068 (2013.01) [C12N 2501/11 (2013.01); C12N 2501/115 (2013.01); C12N 2501/117 (2013.01); C12N 2501/13 (2013.01); C12N 2501/148 (2013.01); C12N 2501/155 (2013.01); C12N 2501/415 (2013.01); C12N 2513/00 (2013.01); C12N 2533/52 (2013.01); C12N 2533/54 (2013.01); C12N 2533/70 (2013.01)] 9 Claims
 
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.
 
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.