A three-dimensional in vitro liver model: Proteomics investigation of cellular adaptations in human hepatic spheroids

Medicine cures, alleviates, and prevents the progression of diseases and as medical science evolves so doesthe opportunity to treat diseases. Drug development, however, requires stringent control to prevent drugswith severe adverse effects from being utilized. This is ensured through clinical trials, which is both timeconsuming and costly. Less than 10% of initiated clinical trials result in drug approval and resources investedin the large number of failed drugs ultimately result in increased medicine prices and slow development ofnew treatments. This could be alleviated by improved pre-clinical screening procedures, which is currentlycarried out in a combination of cell culture and animal models. Animal models, however, suffer from poorinterspecies translatability and conventional cell culture techniques poorly recapitulate in vivo functionalityof organs. We hypothesize that the development of an advanced liver model could improve the predictivepower of pre-clinical screenings.

The liver plays a key role in drug development due to its modification and elimination of drugs, which directly impacts the efficacy of medicines and makes hepatotoxicity a common side effect of many drugs. Hepatocytes are the most abundant cell type in the liver and the primary producers of the enzymes involved in drug metabolism. There are a few different sources of hepatocytes used for in vitro liver models, but immortal cell lines are highly reproducible, easily available, and easy to work with, which makes them ideal for pharmacological applications. 3D cell culture techniques are capable of improving the ability of hepatic
cell lines to recapitulate liver function. The observed benefits of 3D cell culture are believed to be caused by development of microenvironment that closely resembles in vivo conditions.

The aim of this study was to investigate cellular adaptations in long-term cultures of hepatocyte-based spheroids to understand how 3D culture systems improve cellular behavior. In this thesis, I investigate the cellular adaptations that occur within a 3D cell culture model, made from the human hepatocyte cell line
HepG2/C3A, with mass spectrometry-based proteomics. In order to influence the development of extracellular matrix, I introduced another liver cell line, LX-2 cells, to the model and investigated changes in the organization of the cellular microenvironment. The results showed that necrotic core development within spheroids is characterized by core-formation followed by establishment of a stable core. Live cells within stable core-containing spheroids showed an increase in proteins related to the formation of the cellular microenvironment. Further results indicated that 3D co-cultures, containing multiple hepatic cell lines, had increased production of- and interactions with the cellular microenvironment as well as improved liver function in long-term culture.