The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.

Department Leibniz Research Laboratories for Biotechnology and Artificial Organs Hannover Medical School Hannover Germany

Department of Chemistry University of Oslo P O Box 1033 Blindern NO 0315 Oslo Norway

Department of Engineering Faculty of Science Durham University Durham DH1 3LE United Kingdom

Department of Forensic Sciences Oslo University Hospital Oslo Norway

Department of Genetics Yale Stem Cell Center Child Study Center Yale School of Medicine New Haven USA

Department of Haematology Oslo University Hospital Oslo Norway

Department of Histology and Embryology Faculty of Medicine in Hradec Králové Charles University Hradec Králové Czech Republic

Department of Immunology University of Oslo and Oslo University Hospital Oslo Norway

Department of Medical Biophysics Faculty of Medicine in Hradec Králové Charles University Hradec Králové Czech Republic

Department of Medicine Faculty of Medicine Maisonneuve Rosemont Hospital Research Center University of Montreal Montreal Canada

Department of Molecular Medicine Institute of Basic Medical Sciences University of Oslo Oslo Norway

Department of Optical and Biophysical Systems Institute of Physics of the Czech Academy of Sciences Prague Czech Republic

Department of Pediatric Research Oslo University Hospital Oslo Norway

European Reference Network RARE LIVER Hamburg Germany

Hybrid Technology Hub Centre of Excellence Institute of Basic Medical Sciences University of Oslo Oslo Norway

Institute of Clinical Medicine Faculty of Medicine University of Oslo Oslo Norway

Mimetas Leiden The Netherlands

Norwegian PSC Research Center Department of Transplantation Medicine Oslo University Hospital Oslo Norway

Research Institute of Internal Medicine Oslo University Hospital Oslo Norway

Section for Gastroenterology Department of Transplantation Medicine Oslo University Hospital Oslo Norway

Servicio de Hematología y Oncología Médica Hospital Universitario Morales Meseguer Centro Regional de Hemodonación Universidad de Murcia IMIB CIBERER Murcia Spain

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Liver Organoids: Recent Developments, Limitations and Potential