Whole Human liver decellularisation-recellularisation for future liver transplantation and extracorporeal device application

Background and Aims: An estimated 29 million people in the European Union (EU) suffer from a chronic liver condition, with liver transplantation still remaining the only treatment for end-stage hepatic disease. Currently, there are approximately 6700 people awaiting liver transplantation in the EU. Considering that 15- 25% of donated organs are discarded, whole human liver regeneration represents a novel approach to overcome current organ shortages. One possible approach is the use of native extracellular matrix (ECM) as a suitable environment for cells to restore tissue function. Therefore, the aim of this project was to demonstrate, for the first time, the recellularisation of a decellularised whole human liver for future transplantation and extracorporeal device applications. Method: A human liver explant, diagnosed with Crigler–Najjar syndrome, was decellularised using a well- established method, previously characterized for cellular material elimination and preservation of ECM proteins and micro-architecture. Temperature, pH, oxygen and pressure sensors were incorporated into the Harvard Apparatus’ ORCA system, as well as compressed air, O 2 and CO 2 reservoirs. Whole human liver scaffolds (840g) was recellularised by IVC infusion with 2x10 9 HepG2. The liver was maintained in 6 L of complete media with a flow-rate of 400ml/min. The media was changed by replacing 3L of existing media with fresh complete media after 48 hours. The experiment was stopped after 72 hours and the liver was fixed in 4% formaldehyde. The liver was sectioned into 21 parts to investigate repopulation by H&E stating. Albumin secretion was measured using an ELISA kit at 0, 24 and 72 hours. Results: Histological analysis using H&E staining showed that cells have infiltrated all liver segments, excluding segment one. HepG2 cells were seen microscopically to have been migrating from the central vein towards the portal triad, including penetrating into the parenchymal space. Oxygen consumption during the course of three days decreased from 20% to 10%. Additionally, pH was reduced by 0.4. Finally, albumin present in the media increased from 0 ng/ml on day 0, to 200 ng/ml on day 1, to 1500 ng/ml on day 3. Conclusion: This is the first report describing the recellularisation of whole human liver ECM scaffolds with a human hepatocyte cell line. This is a key advance in the development of a bioengineered human liver for future liver transplantation and extracorporeal device applications.