Impact of biomaterials and 3D culture on the hepatocyte phenotype

The liver is one of the most important organs in the human body. It is responsible for a wide variety of metabolic functions. This makes that liver failure is associated with a high mortality. Although the liver has the unique characteristic of regeneration in case of injury, too extended or chronic injury often leads to a life threatening situation for the patients. In this case the only valid treatment is liver transplantation, which is associated with many problems for the patient. With the current shortage of donor organs, many patients die while waiting for transplantation. Alternative treatments such as hepatocyte transplantation or the development of bio-artificial liver devices are hampered by the difficulty to maintain the hepatocyte phenotype when isolated from the liver context. To maintain or prolong the hepatocyte phenotype many techniques have been addressed. The goal of this study was to explore the possibilities of creating a cell-laden 3D construct with predefined dimensions, applying the Bioplotter® technology for hepatic tissue engineering purposes. As such purpose-driven biomaterial development, cultivation in three dimensions and controlling construct configuration was combined in one approach. Gelatin, as starting material, was chemically modified with crosslinkable side groups. This modification enables the creation of a stable hydrogel under physiological conditions. In a first part of the study, methacrylamide-modified gelatin (10 w/v %) was selected as optimal for the encapsulation of single HepG2 cells. Galactosylation of this material had no influence on the cell viability while it increased the expression of hepatocyte specific markers when single HepG2 cells were encapsulated. Encapsulation of single primary hepatocytes however, resulted in an extreme bad cell survival making the encapsulation of single cells an unsuitable approach for primary hepatocytes. As a consequence, the encapsulation of preformed cell aggregates was considered. An agarose microwell chip was used to obtain many uniform HepG2 and primary hepatocyte aggregates of high quality. The formation of aggregates had a positive influence on the hepatocyte phenotype and an optimal aggregate dimension could be selected. The selected aggregates were subsequently encapsulated in the methacrylamide-modified gelatin resulting in a good cell survival and stable hepatocyte phenotype for both the HepG2 cells and primary hepatocytes. Unlike encapsulation of single cells, galactosylation of the material did not influence the hepatocyte phenotype of encapsulated HepG2 or primary hepatocyte aggregates. In a last part of the study single HepG2 cell-laden 3D constructs were generated with the Bioplotter® application. After careful adaptation of the plotting parameters, highly viable HepG2 cells capable of displaying their normal cell behavior were obtained. Recent results have shown that, after some adaptations, similar 3D constructs containing preformed aggregates could be created with the Bioplotter®. This indicates that the creation of 3D constructs containing primary hepatocytes is just one small step away. In conclusion, this study has demonstrated the possibilities of methacrylamide-modified gelatin for the construction of cell-laden constructs with predefined dimensions. This study can apply as a starting point for more advanced hepatic tissue engineering using primary hepatocytes