Production and Characterisation of Acellular Human Liver Matrix: Potential Auxiliary Liver Graft

Shortage of donor organs combined with ever increasing liver disease has directed attention towards growing implantable liver tissue in the laboratory setting. Recent developments in decellularisation and recellularisation techniques have opened exciting facets for liver whole organ reengineering. The aim of this study was to develop a decellularisation protocol for human liver, assess the structural and biochemical integrity and assess the biocompatibility of the acellular matrix. Discarded human livers were split and the left lateral and right posterior lobes with a mean weight of 399 ± 162 g were used (n=8 lobes). Perfusion decellularisation was carried out through the hepatic artery and portal vein. Hypotonic buffer, 0.1 % (w/v) sodium dodecyl sulphate, hypertonic buffer and nuclease solutions were used to remove cells plus nuclear material. Following several iterations of a previously patented Leeds University decellularisation protocol, greater than 91.0 % (w/w) DNA was removed from all areas of liver tissue. Fluoroscopy confirmed integrity of vascular tree. Histological analysis demonstrated lack of cells and maintenance of the portal triad histioarchitecture. Immunohistochemistry demonstrated positive staining for key extracellular proteins such as collagen type I and III, fibronectin and laminin. Scanning electron microscopy showed an intact extracellular matrix. Biochemical assessment confirmed significant reduction in glycosaminoglycan content, but there was increase in collagen content, the latter likely due to extraction by dry weight of other soluble components in the matrix. The acellular tissues and extracts were not cytotoxic to either murine 3T3 or baby hamster kidney cells. Preliminary recellularisation (n=3 lobes) showed the matrix to be biocompatible to primary cryopreserved human hepatocytes seeded at 30,000 cells. The study has developed a suitable protocol for decellularisation of human liver lobes, without adversely affecting the extracellular matrix. The biocompatible acellular scaffold has the potential to be recellularised with hepatocytes, with a view to engineering a transplantable auxiliary liver graft.