Title : Development and characterisation of a Porcine Liver Scaffold . for Clinical Application Running title : A tissue engineered liver scaffold

The growing number of patients requiring liver transplantation for chronic liver disease cannot be currently met due to a shortage in donor tissue. As such, alternative tissue engineering approaches combining the use of acellular biological scaffolds and different cell populations (hepatic or progenitor) are being explored to augment the demand for functional organs. Our goal was to produce a clinically relevant sized scaffold from a sustainable source within 24 hours, whilst preserving the extra cellular matrix (ECM) to facilitate cell repopulation at a later stage. Whole porcine livers underwent perfusion de-cellularisation via the hepatic artery and hepatic portal vein using a combination of saponin, sodium deoxycholate (SOC) and deionised water washes resulting in an acellular scaffold with an intact vasculature and preserved ECM. Molecular and immuno-histochemical analysis (collagen I and IV and laminin) showed complete removal of any DNA material, together with excellent retention of glycosaminoglycans and collagen. FTIR analysis showed both absence of nuclear material and removal of any detergent residue, which was successfully achieved after additional ethanol gradient washes. Samples of the de-cellularised scaffold were assessed for cytotoxicity by seeding with porcine adipose derived mesenchymal stem cells in vitro, these cells over a 10 day period showed attachment and proliferation. Perfusion of the vascular tree with contrast media followed by CT imaging showed an intact vascular network. In vivo implantation of whole intact non-seeded livers, into a porcine model (as auxiliary graft) showed uniform perfusion macroscopically and histologically. Using this method, it is possible to create an acellular, clinically sized, liver scaffold with intact vasculature in less than 24