Establishing species-specific 3D liver microtissues for repeat dose toxicology and advancing in vitro to in vivo translation through computational modelling

The scientific basis of xenobiotic safety is complicated because of the variance in predictability of the primary and secondary pharmacology of foreign chemical substances, as well as variability in individual susceptibility within the population [1]. Despite a wealth of research into this field, our understanding of the mechanisms underpinning the occurrence of adverse effects from xenobiotics remains limited [2]. Adverse drug reactions (ADRs) represent a major encumbrance to the development of new therapeutics with approximately 21% of drug attrition attributed to toxicity during the development process [3]. The in vivo/ex vivo use of animals in science, and in particular drug development, is a global practice and the main purposes of animal experiments are; (i) to gain basic biological knowledge, (ii) for fundamental medical research, (iii) for the discovery and development of drugs, vaccines and medical devices, and (iv) for the toxicity testing of xenobiotics/drugs [4]. However, with there being species-species differences in mechanistic responses, it is difficult to assess results in animal trials and translate these findings in order to predict the in-vivo response in humans [5]. Current in vitro model systems developed to assess ADRs have a number of down falls including; (i) the isolating procedure of primary hepatocytes, (ii) their cost, (iii) inter-donor differences, (iv) limited availability, (v) as well as increasing ethical pressure to implement the 3R’s (Replacement, Reduction and Refinement) in research [6]. The emphasis on producing a relevant and representative in vitro model for hepatotoxicity has therefore expanded. The aim of this thesis is to characterise a novel 3D microtissue model that, in the future, aims to provide a better in vitro platform to assess liver toxicity after repeat-dose exposure to xenobiotics. This is particularly important because the processes of hepatotoxicity manifest themselves over several hours and even days, and therefore in vitro models need to be able to comprehensively assess toxic potential for repeat-dose scenarios as well as chronic exposures. Computational modelling is implemented to 14 allow translation of results and to better bridge the gap between in vitro and in vivo approaches and to exploit the knowledge gained from experimental work. Chapter 1 is a critical review of culture techniques and cell types that are used during the development stages of xenobiotic discovery. A number of in vitro models are evaluated with regards to the determination of hepatotoxic potential of compounds. This review has been previously published [7]. Chapter 1 also includes an introduction to mathematical modelling of hepatic clearance and other pharmacokinetic approaches. Chapter 2 describes the experimental characterisation of a primary rat hepatocyte (PRH) spheroid model. The application of the liquid-overlay technique (LOT) [8] with PRH results in the production of viable and reproducible microtissues, amenable for high-throughput investigations. I show that our in vitro system mimics the in vivo cellular morphology, exhibiting both structural and functional polarisation, along with active and functional transporters. Chapter 3 describes the construction of a mathematical model of oxygen diffusion for my experimental in vitro spheroid system. This model is utilised to predict oxygen profiles within the spheroids and to propose optimised operating conditions in order to recapitulate healthy sinusoidal oxygen tensions. This optimisation is based on initial cell seeding densities and experimentally derived oxygen consumption rates (OCR). Chapter 4 describes the construction of a mathematical model to predict the diffusion of xenobiotics based on their inherent physicochemical properties. The in silico system incorporates specific parameters from the experimental spheroid system including paracellular transport features, namely tortuosity and pore fraction properties. The model describes how these spatiotemporal characteristics vary over the duration of the culture period and what effect these have on the transport of xenobiotics.