Liver-based in vitro models for toxicity testing (#6)
1 Vrije Universiteit Brussel, In vitro Toxicology, Brussels, Belgium
Because of its unique location and function in the organism, the liver is a frequent target for systemic toxicity. Therefore, a lot of attention has been paid and is still being paid to the establishment and further optimization of liver-based in vitro models that can assist in predicting hepatotoxic potential of chemicals. As such, they can be divided in 2 main classes, namely liver-derived in vitro models and stem cell-derived in vitro models. The former include whole isolated perfused livers, liver slices, isolated hepatocytes, liver cell lines and subcellular liver fractions. These liver-derived in vitro models will be discussed in the first part of this presentation, including an overview of their advantages, disadvantages and applications. Focus will be put on primary hepatocytes and their cultures, which are considered as the gold standard in the field of liver-based in vitro modelling. In particular, strategies to increase their longevity and hence to maintain their functional differentiated phenotype in culture will be presented. In the second part of the presentation, a brief overview of stem cell-derived in vitro models will be provided, namely hepatocyte-like cells differentiated from human adult stem cells or human embryonic stem cells by exposure to specific cytokines and growth factors as well as human induced pluripotent stem cells.
Keywords: Liver, In vitro model
Kidney-based in vitro models for toxicity testing (#803)
1 Vrije Universiteit Amsterdam, Division of Molecular and Computational Toxicology, Amsterdam Institute for Molecules, Medicines and Systems, Amsterdam, Netherlands
Each kidney consists of approximately 1 million nephrons at birth. De novo nephrogenesis does not occur after birth and there is no strong evidence suggesting that the adult kidney harbours resident stem cells. Thus, the kidney unlike the liver is not a highly rejuvenative organ, which is most likely an evolutionary compromise allowing the anatomical complexity required for high function and the maintenance of the very narrow margins required for whole body homeostasis. Indeed, the kidney is an extremely accomplished organ and can carry out 100 % of its duties with only a fraction of the nephrons we are born with. However, we continually lose nephrons through-out life and will, all things being equal, eventually breach the renal functional reserve and enter end-stage-renal disease. Thus, anything that contributes to chronic renal failure has the potential to seriously curtail life quality and life-span. Aging populations and associated risk factors such as diabetes and heart disease, have pushed chronic kidney disease (CKD) incidence to unprecedented levels (currently at 10 % of the European population). Due to the role of the kidney in elimination of waste products, renal cells, particularly the proximal tubule, will internally process the majority of drugs and chemicals, and thus often have higher concentrations of these compounds than any other cell in the body. Compounds that injure renal epithelial cells can initiate and/or accelerate CKD. In this lecture, I will discuss the various human in vitro models employed in in vitro nephrotoxicity testing, including primary cells, cell lines and induced pluripotent stem cell models.
Lung-based in vitro models for toxicity testing (#474)
1 Epithelix, Plan-les-Ouates, Switzerland
The main function of the human airway epithelium is to generate sterile atmosphere in the alveolar region where the gas exchange occurs. As the first line of defence against airborne pathogens, the airway epithelium acts as key barrier through mucociliary clearance and innate immune defence mechanisms. Airway epithelium is also an important immuno-regulator through production of key messengers and physical interactions with immune cells. Upon activation, respiratory epithelial cells react by producing pro-inflammatory cytokines, chemokines and metalloproteinases to recruit and activate immune cells such as neutrophils, basophils, or to initiate the adaptive immunity via dendritic cells. Interest in the use of 3D reconstituted human in vitro tissues (ALI cultures) is increasing in recent years for the study of respiratory diseases such as Asthma, Chronic Obstructive Pulmonary Disease (COPD), Bacterial and viral infections, etc.
Genetic and epigenetic diversity in ALIs with single donors allow stratification and patient specific profiling in toxicology and drug testing. On the other hand, ALIs generated with a mixture of cells from several individuals give a snapshot of global reaction of a small population when exposed to a chemical compound.
This talk will describe the in vitro upper and lower respiratory tract models currently available to simulate the human lung epithelial tissue barrier. Several applications of in vitro reconstituted ALI models in inhalation toxicity assessment will be discussed: (i) acute, long-term and chronic testing strategies including static or dynamic exposures; (ii) mucosal inflammation assessment; (iii) trans-epithelial permeability of xenobiotics; (iv) impact on mucociliary clearance and (v) goblet cell hyperplasia monitoring.
Keywords: inhalation toxicity, airway epithelium, Lung toxicity, in vitro lung models
Stem cell-based in vitro models: state-of-the-art tools to predict drug-induced tissue injury in humans (#101)
J. De Kock1
1 Vrije Universiteit Brussel, In vitro Toxicology, Brussels, Belgium
The fact that the detection of drug toxicity is often not accurate and frequently occurs only late during the drug development process is of major concern for the pharmaceutical industry and jeopardizes the potential marketing of new chemical entities. Besides affecting human health, this also leads to a significant loss of resources and time for the pharmaceutical industry. One of the major reasons for this failure is that the safety of new potential drugs is still evaluated in animals or animal-based cell lines. However, efforts at improving the predictability of drug-induced tissue injury in humans are rising due to the rapidly advancing field of human stem cell research. Human target cells for screening and assessment of adverse drug effects in humans can now be generated using pluripotent and multipotent stem cells as detailed in vitro differentiation protocols are established for almost all cell (sub) types. The introduction of state-of-the-art gene editing tools such as CRISPR/Cas9 in stem cell research provides a technology platform to mimic specific genetic polymorphisms or diseases in a dish. In addition, stem cell-based in vitro models can be used to evaluate adverse drug effects during the differentiation process thereby mimicking human developmental toxicity. The latter provides a major advantage over any other non-stem cell-based model. Ultimately, it will be possible to develop personalized toxicology to determine inter-individual susceptibility to adverse drug reactions at any stage of development and in health and disease.
Keywords: stem cells, genome editing, human toxicology, disease-in-a-dish, polymorphism, drug-induced tissue injury
Three-dimensional models for in vitro toxicity testing (#792)
A. C. Gutleb1
1 LIST, ERIN, Belvaux, Luxembourg
Complex 3D in vitro coculture systems are valuable tools to study biological processes at biological barriers or in a tissue in 3D orientation in a more realistic way than in the classic mono-cell type and mono-layer systems. Such 3D systems allow for example direct cell-cell interaction, may allow for cell migration, etc. that all may affect cellular responses. Existing 3D in vitro cocultre models have been established based on stemcells, primary cells and cell lines using transwell inserts or 2D and 3D scaffolds that again influence the biological responses. Overall such models are expected to provide responses more closely resembling effects observable in vivo. In combination with modern systems biology approaches 3D in vitro co-culture systems are the next step to further replace in vivo experiments for example in drug development, hazard assessment and mechanistical toxicity studies as these models allow to follow transport across barriers, cell-cell communication but also to study indirect exposure effects.
Keywords: 3D in vitro models, organoids, co-culture
Beyond the Chip: Development and Application of a Micro-Physiological Flux Analyzer (#817)
Y. Nahmias2, 1
1 Tissue Dynamics, Jerusalem, Israel
Organ-on-chip technology aims to replace animal toxicity testing, but thus far demonstrated few advantages over traditional methods. Current methods to evaluate toxicity rely on end-point assays measuring tissue damage and cell death, resulting in limited kinetic and mechanistic information. We present the Tissue Dynamics platform capable of maintaining vascularized 3D liver, cardiac, and neural tissues for over a month in vitro. Tissues acquire physiological structure, physiological activity and show complex metabolic zonation. Tissue-embedded metabolic sensors for oxygen, glucose, lactate and glutamine permit the real-time quantification of intracellular fluxes and tissue level function. Change in metabolic function is the first indication of physiological stress, preceding any detectable damage. Using the Tissue Dynamics platform, we show a new CYP450-idependent mechanism of acetaminophen toxicity that may be responsible for clinically observed nephrotoxicity. We also show that troglitazone, a drug withdrawn from the market due to idiosyncratic toxicity, induces harmful metabolic changes at below the observed threshold for toxic damage. These metabolic changes may underlie troglitazone’s observed idiosyncratic toxicity. More recently, we studied the dynamics of human liver response to the epilepsy drug valproate. Our platform demonstated a rapid disruption of metabolic homeostasis below the threshold of cellular damage, and an increase in lipogenesis rather than distruption of beta-oxidation. Our work marks the importance of tracing function in real-time, demonstrating specific advantages in predicative toxicology.
Keywords: Organ on chip, idiosyncratic toxicity, liver