Novel approaches for culturing hepatocytes for drug testing applications

Dissertation presented to obtain the Ph.D degree in Biochemisry, BiotechnologyDrug Development is a long and costly process that starts with thousands of drug candidates and ends with one compound, which often fails in clinical trials. This high percentage of failure of the drug development process results from the lack of highly predictable models in the pre-clinical tests. Being metabolism a bottleneck in in vitro drug testing, special efforts have been made towards the development of metabolic competent liver cell cultures. The major drawback of standard hepatocyte cultivation systems is the spontaneous cell dedifferentiation that results in the decrease of metabolic competency and other liver specific functions and ultimately will compromise the effective biotransformation of drugs. Therefore, novel culture strategies presenting an improved hepatocyte viability and functionality for extended periods are required. Within this context, the main goal of this PhD project was to develop an efficient strategy for culturing hepatocytes as 3D spheroid structures since this better mimics the in vivo tissue. Within these 3D structures, cells can re-establish cell-cell interactions and specific microenvironments resulting in a better retention of important hepatic functions. The work here depicted has focused on the use of primary cultures of hepatocytes, since they better retain the liver functional profile. Additionally, the developed strategy has been applied to a human hepatocarcinoma cell line with the aim of generating a more physiological model with cells that are easier to obtain then fresh isolated hepatocytes. Within this context, and also to confirm the robustness of the 3D culturing strategy, three relevant cellular models were used namely, human hepatocytes, rat hepatocytes and the HepaRG cell line.(...)Financial support provided by Fundação para a Ciência e Técnologia (SFRH /BD / 37102 / 2007