Towards graft engineering using decellularized porcine liver scaffolds and recellularization with human liver organoids

J. Willemse, M.M.A. Verstegen, L.J.W. van der Laan, J. de Jonge

Thursday 15 march 2018

12:20 - 12:30h at Van Weelde Zaal

Categories: Basic / translational research, Session (parallel)

Parallel session: Parallel session 2: Basic / translational research

Liver transplantation is the only effective treatment for end-stage liver disease, but donor shortage remains a limiting factor. Recent advances in tissue engineering focus on generation of native extracellular matrix (ECM) scaffold material by decellularizing complete livers. Decellularizing liver tissue is feasible, however, in order to create transplantable liver grafts, these scaffolds need to be recellularized with functional cells such as hepatocytes, cholangiocytes and endothelial cells. Aim of this study is to optimize the recellularization in segments of decellularized porcine liver using hepatic cell lines and human liver-derived LGR5+ organoids.

Porcine livers were obtained from deceased pigs (average weight: 32kg, n = 4) and stored at . Livers were thawed and perfused via the portal vein (average pressure of 90mm Hg), eight hours with 4% triton-x-100 and 1% ammonium solution. Afterwards the liver was washed with demineralized water for 4 hours and DNase-1 (400u/mg) for 2 hours. Histological analysis and DNA quantification confirmed decellularization.

Small liver segments were prepared (average size) and they were cannulated via the arteriole. Segments were incubated () with cell culture medium prior to infusion of cells. Segments are recellularized with HepG2 or human liver organoids. Per segment, 10 million cells are infused in a staged manner. Small volumes 0,5mL, containing 2 million cells, are infused the cannula every 30 minutes, which was repeated for five times. Four hours after the last injection, the segment was connected to a perfusion setup. The segments were perfused for 5 to 7 days with a pulsatile flow of 11mL/min. After the culture period, segments were fixed in 4% paraformaldehyde, processed for paraffin embedding, sectioned and histologically analyzed.

The infused HepG2 and human liver-derived organoids were well capable of infiltrating small pieces of porcine liver scaffold. Cells were found in the parenchymal areas of the liver segments. Small islands could be found in throughout the segments. The organoids self-organized into folded single layered structures with cuboidal cells.

In conclusion, this proof-of-concept study show that recellularization of porcine liver scaffold with human liver- organoids is feasible. These initial results encourage further detailed analysis of the use of decellularized liver ECM as a scaffold material for engineering functional liver tissue.