Many tissue models have been developed to mimic liver-specific functions for metabolic and toxin conversion in in vitro assays. Most models represent a 2D environment rather than a complex 3D structure similar to native tissue. To overcome this issue, spheroid cultures have become the gold standard in tissue engineering. Unfortunately, spheroids are limited in size due to diffusion barriers in their dense structures, limiting nutrient and oxygen supply. Recent developments in bioprinting techniques have enabled us to engineer complex 3D structures with perfusion-enabled channel systems to ensure nutritional supply within larger, densely-populated tissue models. In this study, we present a proof-of-concept for the feasibility of bioprinting a liver organoid by combining HepaRG and human stellate cells in a stereolithographic printing approach, and show basic characterization under static cultivation conditions. Using standard tissue engineering analytics, such as immunohistology and qPCR, we found higher albumin and cytochrome P450 3A4 (CYP3A4) expression in bioprinted liver tissues compared to monolayer controls over a two-week cultivation period. In addition, the expression of tight junctions, liver-specific bile transporter multidrug resistance-associated protein 2 (MRP2), and overall metabolism (glucose, lactate, lactate dehydrogenase (LDH)) were found to be stable. Furthermore, we provide evidence for the perfusability of the organoids’ intrinsic channel system. These results motivate new approaches and further development in liver tissue engineering for advanced organ-on-a-chip applications and pharmaceutical developments.

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.

Background: Ex vivo liver perfusion has emerged in the last years as a promising tool to maintain organ functions and complexity. Hence, it is not only of high importance for transplantation research but also as device or drug testing model. Here, we investigated the possible use of slaughterhouse material to develop a normothermic ex vivo liver perfusion model with an animal friendly approach. The strict processes in a slaughterhouse present a challenging environment for organ procurement and a critical starting state of the organ. Methods: Livers from two slaughterhouses (group 1 and 2) with different slaughtering procedures were compared to lab animal livers. Our developed ex vivo platform was used to reperfuse the livers with physiological pressures at 39°C for 5h. To assess functionality an ICG assay was performed. Further, blood gases, bile production, and gene expression were analyzed to test for viability and damage markers. Results: Physiological flow rates were achieved in all livers. Significant decreases were seen in bile production and functionality for group 1. However, ICG functionality test, bile production, and viability analyses showed comparable results for group 2 to livers from lab animals. Conclusion: This study showed the applicability of slaughterhouse material but indicated an important impact of different slaughter methods on liver performance. Further research will focus on minimization of harmful factors in the slaughterhouse and reconditioning livers before reperfusion, to enable a consistent model independent of slaughterhouse availability. This model allows device testing, organoid implantation and validation of new artificial organs with an animal friendly approach.