To replicate functional liver tissue in vitro for drug testing or transplantation, 3D tissue engineering requires representative cell models as well as scaffolds that not only promote tissue production but also are applicable in a clinical setting. Recently, adult liver‐derived liver organoids are found to be of much interest due to their genetic stability, expansion potential, and ability to differentiate toward a hepatocyte‐like fate. The current standard for culturing these organoids is a basement membrane hydrogel like Matrigel (MG), which is derived from murine tumor material and apart from its variability and high costs, possesses an undefined composition and is therefore not clinically applicable. Here, a cellulose nanofibril (CNF) hydrogel is investigated with regard to its potential to serve as an alternative clinical grade scaffold to differentiate liver organoids. The results show that its mechanical properties are suitable for differentiation with overall, either equal or improved, functionality of the hepatocyte‐like cells compared to MG. Therefore, and because of its defined and tunable chemical definition, the CNF hydrogel presents a viable alternative to MG for liver tissue engineering with the option for clinical use.

In Europe alone, each year 5500 people require a life‐saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large‐animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte‐ and cholangiocyte‐like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large‐animal model.

Metabolic and toxic liver disorders, such as fatty liver disease (steatosis) and drug-induced liver injury, are highly prevalent and potentially life-threatening. To allow for the study of these disorders from the early stages onward, without using experimental animals, we collected porcine livers in a slaughterhouse and perfused these livers normothermically. With our simplified protocol, the perfused slaughterhouse livers remained viable and functional over five hours of perfusion, as shown by hemodynamics, bile production, indocyanine green clearance, ammonia metabolism, gene expression and histology. As a proof-of-concept to study liver disorders, we show that an infusion of free fatty acids and acetaminophen results in early biochemical signs of liver damage, including reduced functionality. In conclusion, the present platform offers an accessible system to perform research in a functional, relevant large animal model while avoiding using experimental animals. With further improvements to the model, prolonged exposure could make this model a versatile tool for studying liver diseases and potential treatments.