Establishing an effective repopulation of the endothelialized liver matrix with primary hepatocytes

• Applicant: Dr. Philipp Felgendreff
• Subject Area: General and Visceral Surgery
• Term: from 2021 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 455452355

Background:Organ engineering is an experimental approach to address the shortage of donor organs. The first step is to generate an acellular scaffold by decellularization, followed by the second step of repopulation with cells and the third step: transplantation of a functional organ.Perfusion with tensides has become the widely accepted method for decellularization. Up to now, liver cell lines, endothelial cell lines, primary hepatocytes, and mesenchymal stromal cells have been used for the reseeding procedure. One of the greatest successes in repopulation so far is the complete re-endothelialization of a decellularized pig liver scaffold, although without using parenchymal cells. After heterotopic transplantation of the re-endothelialized pig liver scaffold, the maintenance of portal venous perfusion in vivo for up to 20 days was demonstrated. However, no liver scaffold repopulated with endothelial as well as parenchymal cells has been transplanted so far.Objective:The aim of this project is the repopulation of the acellular pig liver scaffold with parenchymal and endothelial cells and the proof of portal venous long-term perfusion after heterotopic transplantation.Hypotheses:We hypothesize that the sequential bidirectional application of hepatocytes and endothelial cells via portal and hepatic vein is of advantage compared to unidirectional application. The bidirectional application mode promotes a higher adherence of the applied hepatocytes in the acellular scaffold, increases proliferation and resumption of cell-specific function in vitro and allows long-term perfusion of the repopulated organ in vivo.Experimental design:To achieve this goal, three work packages (WP) are planned. In WP 1 and WP 2, the effect of the cell application route on cell adherence as well as proliferation and resumption of hepatocyte-specific function in the matrix will be investigated in vitro. In WP 3, the influence of endothelial and parenchymal repopulation on portal venous perfusion of the organ matrix after heterotopic transplantation will be assessed. In WP 1, the number of adherent cells in the matrix is determined indirectly by calculating the difference between the number of applied cells and the number of non-adherent cells in the perfusate. In WP 2, resumption of hepatocyte-specific function (e.g. albumin synthesis) is determined repeatedly in cell culture medium. Cell distribution, proliferation rate as well as the zonal enzyme expression along the sinusoid, is analyzed using histological and immunohistochemical methods at the end of the 4-week culture period. In WP 3, after completion of the long-term perfusion culture, the repopulated organ is transplanted in heterotopic position and the portal venous perfusion is evaluated by computer tomography every 72 hours.Perspective:These experiments are the prerequisite for the repopulation of the biliary tree planned in the next step and for the orthotopic transplantation of the xenogeneic organ.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Scott Nyberg, Ph.D.