PhD defence J. (Jorke) Willemse

Dissertation in short:

Liver transplantation is the only treatment options for patients suffering end-stage liver disease. Unfortunately, there is a shortage of donor organs and up to 20% of the patient might not receive a suitable donor liver in time. Bioengineering functional tissue in vitro could be an alternative solution for the organ shortage. These tissue engineering constructs can be used to study hepatobiliary diseases or for transplantation purposes. Scaffold structures are required to bioengineer tissue in vitro, which are capable of supporting cells. Decellularization is a promising method to obtain tissue-specific acellular scaffolds for regenerative hepatobiliary tissue engineering applications. The extracellular matrix (ECM) of decellularized liver consists of a complex mixture of proteins and proteoglycans. These components are deposited in a highly tissue-specific manner and can influence the behavior of cells. The goal of decellularization is to remove all cells but retain the ECM of the liver, including its architecture.

The liver ECM can subsequently act as an organ-specific, acellular scaffold that can be repopulated with viable cells to create functional liver constructs. Hepatocytes perform a large part of the liver’s functions. These cells metabolizes different substances, but can also produce compounds such as proteins, carbohydrates and bile. The produced bile is drained via the biliary tree. The liver requires protection against bile, since it is cytotoxic. This protective function is performed by cholangiocytes, which can be found in the bile ducts. Other required cell types include endothelial cells (vasculature), hepatic stellate cells and Kuppfer cells.

These cells need to be expanded in vitro before they can be used for repopulation of the empty matrix. Isolation and expansion of hepatocytes and cholangiocyte is challenging and long term expansion using traditional culture methods is nearly impossible. Cholangiocyte organoids are an attractive alternative source of human cholangiocytes. The organoids can be initiated from relative small tissue biopsies, but can yield large amounts of cells because of their high and stable proliferation capacity.

The aim of this thesis is to explore the possibilities of using decellularized liver ECM and cholangiocyte organoids to tissue engineer functional liver tissue in vitro. In this thesis, we described how decellularization protocols for human and porcine livers were set up to obtain scaffold from liver tissue (part 1). Subsequently, cholangiocyte organoids are investigated to repopulate liver ECM (part 1) and extrahepatic bile duct ECM (part 2). In part 3, hydrogel derived from liver ECM are investigated as tissue-specific and clinically relevant culture substrate for the initiation and expansion of organoids.