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Objective: A tissue engineered liver graft will provide a sustainable source of transplantable organs. A porcine liver scaffold was utilized to develop a re-endothelialized revascularized liver capable of sustained in vivo perfusion in a large animal model. Background: Liver transplantation remains the only definitive treatment for end-stage liver disease. With only 6000 donor livers available each year nationally, the need far exceeds the supply. Innovative cell-based therapies, such as hepatic tissue engineering, offer alternatives to supplement the limited supply of quality organs for transplantation. Methods: Whole livers harvested from 6-10 kg pigs underwent rapid perfusion decellularization resulting in an acellular scaffold with an intact vascular network and capsule. Scaffolds were re-endothelialized with Human or Porcine Endothelial Cells derived from Human or Pig Umbilical Vein (HUVEC or PUVEC cells) at physiologic perfusion pressures. PUVEC seeded grafts were implanted as auxiliary liver transplants in recipient pigs. Results: Non-endothelialized grafts demonstrated thrombosis at 30 minutes post perfusion with absent vascular inflow. Re-endothelialized grafts demonstrated patent vascular inflow, absence of gross thrombus, and patent post-explant portal venogram after 72 hours of in vivo hemoperfusion. Histologic review demonstrated viable endothelial cells following graft explant. Conclusions: Vascular patency of a re-endothelialized porcine whole liver graft was achieved in vivo in the absence of systemic anticoagulation. This implantable bioengineered re-endothelialized liver offers early proof of concept of a future unlimited source of patient specific livers for transplantation. *Corresponding author: Scott L Nyberg, MD, PhD, Department of Surgery, William J. von Liebig Center for Transplantation and Clinical Regeneration, Mayo Clinic 200 First Street Rochester, MN 55905, USA, Tel: 507-266-6772, Fax: 507-266-2910, E-mail: Nyberg.scott@mayo.edu ISSN: 2572-4045 DOI: 10.23937/2572-4045.1510031 Mao et al. Int J Transplant Res Med 2017, 3:031 • Page 2 of 9 • the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal care was conducted in accordance with the Mayo Clinic Institutional Animal Care and Use Committee Standards. This specific study was approved under protocols A58912 and A49315. All animals were housed, fed, cared for, and euthanized by study personnel and animal husbandry staff of the Mayo Clinic Department of Comparative Medicine. Procurement of donor liver Donor livers were harvested immediately post-mortem from 10 kg donor pigs obtained from a slaughterhouse, Midwest Porcine Recovery. Animals were sedated and euthanized via lethal direct intracardiac injection of sodium pentobarbital by study personnel. A midline laparotomy was performed immediately following euthanasia. The portal vein was isolated and cannulated with 10 French tubing. The liver underwent in situ perfusion with 1 L of chilled phosphate buffered saline. The hepatic artery and common bile duct were suture ligated. The infrahepatic and suprahepatic IVC were dissected to allow for ample length for post-explant cannulation. The liver was explanted and further perfused with 1 L of phosphate buffered saline and stored at 4 °C. Whole liver decellularization The explanted livers were cannulated and continuously perfused at 12 mmHg throughout the decellularization process. The livers were actively decellularized with 0.6% SDS 4-12 liter baths for up to 48 hours, followed by serial washes in sterile water, 0.1% Peracetic Acid (PAA), and Phosphate Buffered Saline (PBS). To reduce the bioburden risk, the decellularization process was done in a class 10,000 cleanroom using aseptic techniques. Isolation of PUVECs Porcine Umbilical Cord Vein Endothelial Cells (PUVECs) was isolated from unrelated donor animals. Only umbilici from type O animals were used to isolate endothelial cells. To isolate PUVECs, an umbilicus was washed with a solution of 4% penicillin streptomycin in PBS until all visible red blood cells were removed. Vessel branches were ligated, and the vessel lumen filled with a digestion solution consisting of 10,000 U of Type 2 collagenase (Invitrogen) and 0.5 g of BSA (Sigma) suspended in 50 mL of PBS. The vessel was incubated for 15 min at 37 °C. The vessel was massaged to enhance cell release and the cells collected by centrifugation. The digestion was repeated and cultures started independently to isolate those cultures consisting of endothelial cells. Cells were cultured in complete Endothelial Growth Media (EGM-2, Lonza) and passaged at approximately 90% confluence. Endothelial cells were confirmed by cell surface markers (CD-31, manufacture), LDL uptake, and morphology. Cells were grown up in large lots and cryopreserved for re-endothelialization. Prior to re-endothelialization, cells were placed into culture and allowed to fully recover. limited supply of compatible organs for transplantation. Alternative strategies for organ procurement have been trialed, including xenogeneic transplant and cellbased transplantation. Application of xenogeneic organs has been limited due to immune rejection and graft thrombosis. Cell-based hepatocyte transplantation offers another alternative to whole organ transplantation; however, limitations in cell supply, cell quality, and poor engraftment, especially under cirrhotic conditions, have limited the clinical efficacy of cell-based therapy. The use of whole organ tissue engineering overcomes these barriers to engraftment and offers potential for an unlimited supply of transplantable organs. Whole liver tissue engineering utilizes a scaffold or Extracellular Matrix (ECM) to allow for growth and expansion of patient specific cellular material on a background of native organ structure [3]. The use of a porcine decellularized organ matrix preserves the natural ECM proteins and growth factors which allow for cell attachment and proliferation in a native configuration [4]. The presence of an ECM has been demonstrated to support cell mitogenesis and chemotaxis, direct cell differentiation, and support cell remodeling [5]. Prior to the report of perfusion decellularization, decellularized tissues were limited to relatively thin samples (small intestine submucosa, bladder, skin, etc.) [6]. In contrast, whole organ perfusion decellularization has achieved a form of whole organ decellularization that preserves the native three-dimensional structure, mechanical properties, architecture, and vascular network [7,8]. The result is an ideal liver specific scaffold for liver engineering that can be seeded with patient specific endothelial cells, hepatocytes, cholangiocytes, or progenitor cells. This will allow for engineering of a transplantable organ specific to the recipient, circumventing the need for immunosuppression [9]. A critical step in tissue engineering a transplantable liver is the creation of a functional vasculature capable of long-term perfusion following anastomosis. Without an appropriate endothelial lining of the vessels, continuous blood perfusion of the graft in the absence of anticoagulation quickly results in thrombosis. In this study, we report a method for endothelial seeding of a decellularized porcine liver graft and evaluation of seeded grafts under hemoperfused in vivo conditions. The juvenile porcine liver was selected for its anatomical structure similar to that of a human liver. Grafts were seeded in vitro using allogeneic (porcine) umbilical cord derived endothelial cells. The cells provided single layer coverage of vascular structures and the hepatic sinusoids within the graft to reduce the risk of thrombogenesis. We observed no evidence of graft thrombosis in recipient animals after 60 minutes and limited thrombosis at the study endpoint of 72 hours in the absence of chronic anticoagulation.

Sustained In Vivo Perfusion of a Re-Endothelialized Tissue Engineered Porcine Liver