Granted Patent
B2
US 12,649,903 · App. 17/191,650 · Granted Jun 9, 2026
Liver organoid, uses thereof and culture method for obtaining them
C12N5/0671A61K35/407C12N5/0672C12N2500/38C12N2501/11C12N2501/119C12N2501/12C12N2501/155C12N2501/415C12N2533/90
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Abstract
The invention relates to a liver organoid, uses thereof and method for obtaining them.
Claims (53)
1 . An in vitro composition comprising a liver organoid, wherein the organoid comprises a cystic structure comprising an outer layer, which comprises cells, and a lumen, and can be cultured for at least three months,
wherein the outer layer of cells comprises Lgr5+ liver epithelial stem cells, wherein the organoid is obtainable by an in vitro method comprising culturing and expanding Lgr5+ liver epithelial stem cells 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+ liver epithelial stem cells.
2 . The composition of claim 1 , wherein the organoid comprises
(a) a hepatocyte or a cholangiocyte cell, or
(b) a hepatocyte and a cholangiocyte cell.
3 . The composition of claim 1 , wherein the organoid expresses at least one of the following markers: a hepatocyte marker selected from albumin, B-1 integrin, transthyretrin and Glutamine synthetase; and/or at least one cholangiocyte maker selected from Keratin 7 and 19.
4 . The composition of claim 3 , wherein the expression is detected by
(a) measuring the expression at the mRNA level and/or
(b) using a microarray.
5 . The composition of claim 1 , wherein between 25% and 45% of the cells comprised by the organoid express a hepatocyte marker.
6 . The composition of claim 5 , wherein the hepatocyte marker is albumin, B-1 integrin, transthyretrin or Glutamine synthetase.
7 . The composition of claim 5 , wherein 35% of the cells comprised by the organoid express a hepatocyte marker.
8 . The composition of claim 1 , wherein the organoid
(a) comprises a population of between at least 1×10 3 cells and 5×10 4 cells, and/or
(b) has a cell density between 5×10 5 cells/cm 3 and 10×10 5 cells/cm 3 .
9 . The composition of claim 8 , wherein the organoid has a cell density of greater than 10×10 5 cells/cm 3 .
10 . The composition of claim 1 , wherein the layer of cells comprises at least one bud.
11 . The composition of claim 1 , wherein the organoid has an elongated shape with two defined domains.
12 . The composition of claim 11 , wherein the two defined domains comprise
a first domain, wherein the first domain comprises a single layered epithelial domain comprising highly polarized cells, and wherein keratin markers are expressed; and
a second domain, wherein the second domain comprises a main body of the organoid and is formed by a multilayered epithelia comprising non-polarized cells.
13 . The composition of claim 12 , wherein albumin expression is detected in the second domain.
14 . The composition of claim 1 , wherein the organoid
(a) secretes albumin,
(b) secretes urea,
(c) comprises glycogen stores, and/or
(d) has inducible cytochrome P450 activity.
15 . The composition of claim 1 , wherein at least 2% to at least 95% of the cells comprised by the organoid express Lgr5.
16 . The composition of claim 1 , wherein the organoid expresses
(a) at least one or all of the following hepatocyte specific genes: TTR, ALB, FAH, TAT, CYP3A7, APOA1, HMGCS1, PPARG, CYP2B6, CYP2C18, CYP2C9, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F8, CYP4V2 and SCARB1,
(b) expresses at least one or all of the following hepatocyte/cholangiocyte specific genes: HNF1A, HNF1B, HNF4A, HHEX, ONECUT1, ONECUT2, PROX1, CDH1, FOXA2, GATA6, FOXM1, CEBPA, CEBPB, CEBPD, CEBPG, GLUL, KRT7, KRT19 and MET,
(c) expresses at least one or all of the following stem cell signature genes: LGR4, TACSTD1/Epcam, CD44, SOX9, SP5, CD24, PROM1, CDCA7 and ELF3, and/or
(d) expresses at least one or all of the following reprogramming genes: KLF4, MYC, POU5FI and SOX2.
17 . The composition of claim 1 , wherein the organoid does not express
(a) at least one of the following hepatocyte/cholangiocyte specific genes: NEUROG2, IGF1R and CD34, AFP, GCG and PTF1A, or
(b) NEUROG2, IGFIR and CD34.
18 . The composition of claim 1 , wherein the organoid is
(a) a mouse organoid or
(b) a human organoid.
19 . The composition of claim 1 , wherein the composition further comprises an extracellular matrix.
20 . The composition of claim 19 , wherein
(a) the extracellular matrix is an exogenous extracellular matrix, and/or
(b) the extracellular matrix is a non-mesenchymal extracellular matrix.
21 . The composition of claim 19 , wherein the organoid is
(a) embedded in the extracellular matrix, and/or
(b) in contact with the extracellular matrix.
22 . The composition of claim 1 , wherein the organoid
(a) is not a liver fragment,
(b) does not comprise a blood vessel, and/or
(c) does not comprise a liver lobule or a bile duct.
23 . The composition of claim 1 , wherein the layer of cells is 2-30 layers of cells.
24 . The composition of claim 1 , wherein the layer of cells is 2-15 layers of cells.
25 . A pharmaceutical composition comprising the composition of claim 1 for treating a liver disorder, condition or disease or in regenerative medicine.
Assignments (3)
CORRECTIVE ASSIGNMENT TO CORRECT THE APPLICATION NUMBER 15665363 PREVIOUSLY RECORDED AT REEL: 057851 FRAME: 0768. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT .
Recorded Oct 29, 2021
From: ORTEGA, MERITXELL HUCH; CLEVERS, JOHANNES CAROLUS
To: KONINKLIJKE NEDERLANDSE AKADEMIE VAN WETENSCHAPPEN
Reel/Frame 057970/0610 →
CORRECTIVE ASSIGNMENT TO CORRECT THE ASSIGNEE'S ADDRESS PREVIOUSLY RECORDED AT REEL: 57372 FRAME: 552. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT.
Recorded Oct 8, 2021
From: ORTEGA, MERITXELL HUCH; CLEVERS, JOHANNES CAROLUS
To: KONINKLIJKE NEDERLANDSE AKADEMIE VAN WETENSCHAPPEN
Reel/Frame 057851/0768 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Sep 2, 2021
From: ORTEGA, MERITXELL HUCH; CLEVERS, JOHANNES CAROLUS
To: KONINKLIJKE NEDERLANDSE AKADEMIE VAN WETENSCHAPPEN
Reel/Frame 057372/0552 →
Priority Claims (1)
EP 10171265
· Jul 29, 2010
· regional
Continuity (5)
Continuation
13812614
Provisional Application
61520569
· Jun 10, 2011
Provisional Application
61368736
· Jul 29, 2010
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