In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.
In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.
Medicyte GmbH, Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.
Sarada Devi
SD
Ramachandran
Department of Medicine II, Faculty of Medicine at Mannheim, Heidelberg University, Mannheim, Germany.
Katharina
K
Schirmer
Medicyte GmbH, Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.
Bernhard
B
Münst
Medicyte GmbH, Im Neuenheimer Feld 581, 69120, Heidelberg, Germany; upcyte technologies GmbH, Osterfeldstraße 12-14, 22529, Hamburg, Germany.
Stefan
S
Heinz
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany.
Shahrouz
S
Ghafoory
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany.
Stefan
S
Wölfl
Institute of Pathology, University Medical Centre Mannheim, Heidelberg University, Mannheim, Germany.
Katja
K
Simon-Keller
Institute of Pathology, University Medical Centre Mannheim, Heidelberg University, Mannheim, Germany.
Alexander
A
Marx
Vascular Biology Research Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Sykehusgt. 44, N-9037, Tromsø, Norway.
Cristina Ionica
CI
Øie
Department of Medicine II, Faculty of Medicine at Mannheim, Heidelberg University, Mannheim, Germany.
Matthias P
MP
Ebert
Tissue Engineering and Regenerative Medicine, University Wuerzburg, Roentgenring 11, 97070, Würzburg, Germany.
Heike
H
Walles
Medicyte GmbH, Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.
Joris
J
Braspenning
Department of Medicine II, Faculty of Medicine at Mannheim, Heidelberg University, Mannheim, Germany.
Katja
K
Breitkopf-Heinlein
D000328:Adult; D000418:Albumins / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D003094:Collagen; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004338:Drug Combinations; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007797:Laminin; D008099:Liver / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D018613:Microscopy, Confocal; D009940:Organoids / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D011509:Proteoglycans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods; D062826:Zonula Occludens-1 Protein / Q000235:genetics / Q000378:metabolism
D000418:Albumins; D015820:Cadherins; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C059485:matrigel; D003094:Collagen; D003577:Cytochrome P-450 Enzyme System