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Background & Aims Hepatitis C virus (HCV) is a leading cause of chronic liver diseases and the most common indication for liver transplantation in the United States. HCV particles in the blood of infected patients are characterized by heterogeneous buoyant densities, likely owing to HCV association with lipoproteins. However, clinical isolates are not infectious in vitro and the relative infectivity of the particles with respect to their buoyant density therefore cannot be determined, pointing to the need for better in vivo model systems. Methods To analyze the evolution of the buoyant density of in vivo–derived infectious HCV particles over time, we infected immunodeficient human liver chimeric fumaryl acetoacetate hydrolase-/- mice with J6/JFH1 and performed ultracentrifugation of infectious mouse sera on isopicnic iodixanol gradients. We also evaluated the impact of a high sucrose diet, which has been shown to increase very-low-density lipoprotein secretion by the liver in rodents, on lipoprotein and HCV particle characteristics. Results Similar to the severe combined immunodeficiency disease/Albumin-urokinase plasminogen activator human liver chimeric mouse model, density fractionation of infectious mouse serum showed higher infectivity in the low-density fractions early after infection. However, over the course of the infection, viral particle heterogeneity increased and the overall in vitro infectivity diminished without loss of the human liver graft over time. In mice provided with a sucrose-rich diet we observed a minor shift in HCV infectivity toward lower density that correlated with a redistribution of triglycerides and cholesterol among lipoproteins. Conclusions Our work indicates that the heterogeneity in buoyant density of infectious HCV particles evolves over the course of infection and can be influenced by diet., Graphical abstract

In spite of the vast collective experience in tissue engineering, control of both tissue architecture and scale are fundamental translational roadblocks. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. Here, we introduce an approach called ‘SEEDs’ (‘in Situ Expansion of Engineered Devices’), in which we fabricate a structurally organized engineered tissue unit that expands in response to regenerative cues after implantation. We find that tissues containing pre-patterned human primary hepatocytes, endothelial cells, and stromal cells in degradable hydrogel expand over 50-fold over the course of 11 weeks in animals with liver injury, with concomitant increased function as characterized by the production of multiple human liver proteins. Histologically, we observe the emergence of stereotypical microstructure via coordinated growth of hepatocytes in close juxtaposition with a perfused, chimeric vasculature. Importantly, we demonstrate the utility of this platform for probing the impact of multicellular geometric architecture on tissue expansion in response to regenerative cues. This approach represents a hybrid strategy that harnesses both biology and engineering to deploy a limited cell mass more efficiently than either approach could do in isolation, and thus offers a new convergent paradigm for tissue engineering.