Formulating an improved in vitro hepatic model for drug development and toxicity testing

Abstract

There is a need in the pharmaceutical industry for more informative and functional in vitro human models for drug testing as the currently used animal models have poor correlation to their human counterparts. The liver is the main organ of metabolism and xenobiotics detoxification. As such, a human hepatic in vitro model with improved metabolic functions similar to primary human hepatocytes (PHHs) could reduce the number of animals needed in pre-clinical testing and enhance the relevance of data obtained for subsequent in vivo testing. Most immortalized hepatic cell lines are derived from carcinomas and do not retain a full range of functional activity in vitro. Here we have compared a novel hepatic cell line (HepaRG™), an intrinsic co-culture of hepatocyte and cholangiocyte-like cells, with the commonly used C3A cell line which is a derivative of HepG2 cells, in terms of metabolic competency. We found that the HepaRG™ cells out-perform C3As in a number of metabolic functions that include phase I and II metabolism as well as CYP activity. This makes the HepaRG™ cell line a strong alternative to PHHs for in vitro pre-clinical drug testing. Next, we considered the platform for in vitro modelling. There are currently many tissue engineering models available with improved cell culture characteristics such as 3D spheroids, microfluidic models or 3D printing. However, these methods are costly and time consuming. We have used nanopatterned culture plates to develop a cheaper and faster platform that will produce an enhanced human hepatic culture capable of sustaining a differentiated state for several weeks. Such a model will also allow for multi-experimentation or repeat dosage and would be a significant step towards reducing small animal in vivo testing and may correlate better to pre-clinical human trials. We have specifically selected a nanopatterned and oxygen plasma treated culture system that has shown promise in differentiating other stem-like cells into organ specific cultures without the use of additional chemicals or hormones. By growing HepaRG™ progenitor (HepaRG-P™ ) cells on these prototype plates, we showed a much earlier differentiation compared with the established HepaRG-P™ cell culture protocols. Improved functionality at this early time point can also be seen in terms of CYP activity and markers of maturation. There is also some evidence to suggest specific zonation of mature HepaRG™s within this model. Finally, a real-time, label–free monitoring of cell culture fitness, that encompasses a quantitative analysis of cell culture during treatment with a pharmacological agent, is desirable. An electrical cell impedance substrate (ECIS) platform that fulfils the above criteria was validated for real-time, non-invasive, monitoring of the HepaRG™ cell culture. Chlorpromazine (CPZ), a model cholestatic drug, was used to assess its effect on the HepaRG™ cells using ECIS. This study also gave us the opportunity for a more in-depth analysis of CPZ-induced cholestasis by not only analysing tight junctions, adhesion and cell membrane integrity, but also by studying the bile acid and xenobiotic transporters and the inflammatory and adaptive responses to CPZ-induced injury. We have shown disruption of membrane integrity, changes in bile acid transporters and the regulation of both xenobiotic and phospholipid transporters as well as inflammatory markers. In conclusion, in this thesis I have demonstrated that by using a novel cell line with novel prototype culture plates (nanopatterned) and a label free technique (ECIS) to measure cellular fitness, a much improved in vitro model of drug testing can be developed.