Functional characterization of a bioengineered liver after heterotopic implantation in pigs

Brett D Anderson,Dongjin Joo,Aleksandr A Katane,Alyssa Mendenhall,R Noelle Palumbo,Scott L Nyberg,Benjamin G Steiner,Dominique S Davidow,Bruce P Amiot,Aron R Stumbras,Erek D Nelson,Thomas W Gilbert,Victoria L Nelson,Jeffrey J Ross

doi:10.1038/s42003-021-02665-2

Abstract

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

Highlights

Organ bioengineering offers a promising solution to the persistent shortage of donor organs
We recently reported the reconstitution of a functional vascular network in a porcine whole liver scaffold with human umbilical vein endothelial cells (HUVECs), which reproducibly sustained in vivo perfusion for an excess of 10 days in a porcine liver transplant model under a steroid-based immunosuppression regimen.[17]
We previously reported that daily glucose consumption rates (GCR) provide a robust, non-invasive metric for monitoring cell proliferation in HUVEC-seeded bioengineered liver (BEL) constructs and is predictive of successful perfusion outcomes following in vivo inplantation[17]

Summary

Introduction

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells. The current study further shows that these BELs are amenable to surgical implantation and continuous perfusion in a large animal heterotopic liver transplant model, with the maintenance of cell viability and functional marker expression 48 h following implantation These studies describe a robust platform for the seeding and culture of multiple liver cell types, and demonstrate proof of concept for bioengineering a therapeutic liver construct at a clinically relevant scale

Methods

Results

Conclusion