*Purpose: Liver transplantation has shown to be an effective therapy for the end-stage liver diseases (ESLD). However, as donor organ shortages increase, organ engineering technology offers an alternative curative option for ESLD. Currently, application of this technology towards clinical use is limited by challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions. Previously, we developed a functionally revascularized liver graft using human umbilical vein endothelial cells (HUVEC’s). These grafts successfully sustained continuous perfusion in a heterotrophic implantation in a large animal model.

*Methods: In this study, we establish methods for seeding and culturing of primary porcine hepatocytes into revascularized liver grafts and characterize these bioengineered livers (BELs) for liver-specific functions and vascular patency in acute blood perfusion studies.

*Results: These BELs demonstrated liver-specific functions such as ammonia clearance, albumin synthesis, and urea production during culture in our in-vitro bioreactor system. Additionally, we evaluated the function and patency of these BELs in a heterotopic BEL implantation surgical model in pigs with portacaval shunt surgery-induced acute liver failure. These BELs slowed ammonia accumulation and remained patent throughout the post-transplantation period in pigs having acute liver failure. Evaluation of pre and post-implantation gene expression showed that these BELs retained canonical endothelial and hepatocyte-specific markers expression.

*Conclusions: These results taken together indicate that these culturing and seeding strategies have generated functional BELs, which can be scaled to clinical use for liver transplantation in the future.

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