*Purpose: Advancements in whole organ bioengineering have the potential to offer solutions to a chronically insufficient donor organ supply. We recently demonstrated an ability to reconstitute a functional vascular network in a decellularized porcine liver scaffold with human umbilical vein endothelial cells. These revascularized constructs were capable of sustaining continuous perfusion in vivo in an immunosuppressed porcine heterotopic transplantation model for up to 15 days. Here, we build on our previous work by seeding revascularized liver constructs with primary porcine hepatocytes. The resulting bioengineered liver (BEL) constructs were characterized for cell distribution, in vitro hepatic function, and vascular patency.

*Methods: Porcine livers were cannulated on all major vessels and perfusion decellularized with a series of detergent solutions to generate liver scaffolds. Human umbilical vein endothelial cells (HUVECs) were seeded into liver scaffolds through the portal and hepatic veins and cultured in perfusion bioreactors for 13-16 days prior to seeding porcine hepatocytes through the bile duct. Cell distribution and functional marker expression were evaluated by H&E staining and immunofluorescence microscopy. Production of albumin and vWF was assayed over the course of bioreactor culture, and ammonia detoxification kinetics were measured following BEL exposure to 0.8 mM ammonium chloride. Vascular patency was assessed in an in vitro blood loop system and ex vivo in a porcine blood perfusion model. The extent of vascular patency was imaged by real-time angiography during active blood perfusion.

*Results: Histological analysis of BELs revealed hepatocyte clustering in parenchymal lobules, while endothelial cells localized primarily within vessels. HUVECs localized within parenchymal capillaries expressed LYVE1, suggesting a microenvironment-dependent transition toward a liver sinusoidal endothelial cell-like phenotype. Production of vWF was observed over the course of BEL culture, and albumin production was detectable in the days following hepatocyte seeding. BELs efficiently cleared ammonia and produced urea following the addition of exogenous ammonia to the culture media. BELs remained patent upon physiologic blood perfusion with significant flow evident throughout the majority of the scaffold.

*Conclusions: In this current study, we demonstrate the feasibility of creating a clinical scale BEL using a decellularized porcine liver scaffold seeded with resident liver cell types. These results described a robust platform for optimizing the seeding of additional liver specific cell types that will enable functional studies in a large animal transplant model to further develop and assess the functionality of a transplantable BEL.

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