The project DILI-on-chip addresses the development of a microfluidic device to assess in vitro adverse drug reactions, on a human-like liver lobule. We will focus on the particular case of DILI (drug induced liver injury). From a unique donor, hiPSCs (human induced pluripotent stem cells) will be differentiated to obtain the different types of cells, hepatocytes as well as non-parenchymal cells, that compose the liver lobule. Hepatocytes forming the hepatic cord, bordered by endothelial cells forming the sinusoid, and cholangiocytes forming the bile ducts will be organized in 3D on the chip, in order to get a functional unit mimicking the Hering channel. Kupffer cells will also be introduced in the vascular channels, as they are involved in the immune aspects of DILI. Multiple hepatic cords will be contained in microfluidic chambers, organized in circular lobule-like structures, and vascularized in order to induce the hepatocyte polarization, and bile canaliculi formation. Bile ducts will be formed in our chip, for the first time, by cholangiocytes maturation at the continuation of the hepatic chamber. Such biliary ways reconstructed on a chip is of prime importance as bile extraction is mandatory for the long term viability required for DILI assays. The microfluidic device will be composed of two parts ; i) a supporting substrate where will be organized the fluidic connections level, with the vascular and biliary flows. This substrate will be made out of Polydimethylsiloxane (PDMS) using the conventional soft lithography techniques or replicated in plastic (PolyMethylMetAcrylate) by hot embossing ii) on the top of this substrate will be assembled cell culture units, made out of polysaccharides gel, that have the suitable stiffness properties near to the one of the liver parenchymatic tissue, and the appropriate porosity for the nutrient and oxygen exchanges. The geometry of the microfluidic circuitry molded in this gel will be optimized in order to induce the tubular self-organization of cholangiocytes and endothelial cells, with the help of specific functionalization of the gel. An important feature of the device is the possible biodegradability of the gel part, that will render possible the collection of cells or cell content, for proteomics or transcriptomic analysis after drug exposure of the organ on chip. The polysaccharide gel structured units will be arrayed on the microfluidic subpart, in order to prove the feasibility of parallelization of the concept, with possible drug screening by the spotting of several drugs at different concentrations on the array, and collection of bile and vascular flows, for toxicology analysis in the context of DILI. Hepatotoxic compounds effects on the organ on chip will be analyzed, characterizing firstly the drug metabolism activity of the cells, then monitoring the damages induced to liver cells. Cumulative mechanisms of DILI will be finally addressed, by the drug exposure to cells presenting pre-existing pathologies. The project is organized in five tasks, including the management task. The tasks 1 will focus of the obtention of the different cells types that will compose the liver on chip, that will be differentiated from Induced pluripotent stem cells (iPSCs) from the same donor. The consortium will benefit from the huge experience of partner 2 in iPSCs cells differentiation. The second task concerns the fabrication of the microfluidic device, combining the expertises of the partners in microfluidics systems fabrication, and in the preparation and patterning of biodegradable gels. Task 3 will be devoted to the cell loading and culture within the device, including the fluidic instrumentation of the experiment. The formation of a functional hepato-cholangiocyte transition will be characterized on the chip in real-time. Finally, the task 4, led by the industrial partner, will focus on the drug assay on the functional liver on chip, in the DILI application context.

Chronic liver diseases affect more than 500 million people worldwide. Liver transplantation is the only treatment for severe diseases, but it is limited to a small number of patients due to the shortage of organs. The implantation of bioengineered liver tissue represents a major hope. To this end, it is necessary to propose an efficient strategy for the production and freezing of tissue-engineered products that can be rapidly available at the patient's bed. We have gathered several internationally renowned academic and industrial partners with a high level of expertise in complementary fields (liver biology, human induced pluripotent stem cells (hiPSCs), organoids, encapsulation in biomaterials, and cryopreservation) to address this multidisciplinary challenge. We propose to develop protocols to freeze and thaw encapsulated organoids formed from hepatocytes, mesenchymal stem cells, and endothelial cells all derived from hiPSCs. Their cryopreservation being the major issue of this project. Different processes have already been identified and will be evaluated. Encapsulation in porous alginate beads will ensure the protection of the organoids during these phases and will facilitate future implantation in the patient. Different cellular functions will be studied in vitro during all production steps and evaluated in a preclinical mouse model. This is the first time that a project integrates all these innovations to meet the healthcare needs of the 21st century in liver regenerative medicine. Indeed, the development of cryopreservation is necessary to allow a complete characterisation of the different batches (safety/efficacy) while ensuring the rapid availability of functional and implantable encapsulated organoids in the liver.

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial chronic inflammatory disease that is prevalent in 1 of 4 individuals with a significant personal, socioeconomic and healthcare burden, especially at the later, more severe inflammatory stage of disease - non-alcoholic steatohepatitis (NASH). Despite the severe negative impact of the disease on society, NAFLD remains difficult to diagnose and treat. Additionally, the molecular mechanisms underlying the transition from health to fatty liver to NASH remain poorly understood due to the lack of models that faithfully reflect the complexity of human disease. Hence, Halt-RONIN aims to uncover the early triggers of disease initiation and complex mechanistic drivers of disease progression by implementing a systems biology approach with integrative disease modelling resulting in opportunities for the improvement of the existing detection methods, providing a blueprint to inform personalized intervention strategies and drug discovery for NAFLD. To achieve this goal, Halt-RONIN will combine experimental data from advanced in vitro and in vivo models with multimodal data from extensive human NAFLD cohorts and biobanks and use in silico machine learning approaches, to discover new biomarkers and molecular targets specific to each stage of the health-to-disease transition. By validating preclinical experimental findings with real-world data, RONIN will allow for the discovery of novel biomarkers and molecular targets that are specific to the individual patient’s pathology. Consequently, healthcare professionals will gain the tools and knowledge required to diagnose and establish guidelines for the prevention and treatment of inflammation-driven health to disease. As such, in the long-term RONIN will decrease the number of NAFL patients who progress into NASH and provide disease-modifying strategies to improve patient outcomes.

Mass-production of high-quality human hepatocytes is at the heart of an economic challenge, biotechnology and biomedical research. Indeed, the liver is the major organ for the destruction and elimination of endogenous metabolic wastes and xenobiotics (pollutants, pesticides, medical drugs). It is responsible for the metabolism of many drugs into active metabolites. However, these active metabolites, or their intermediates may induce liver toxicity. Unexpected problems of toxicity and pharmacokinetics are responsible for most failures in clinical drug development. The liver performs other vital functions such as the production of most of the serum proteins and lipids, bile production, the metabolism of food into nutrients, and glucose homeostasis (storage). Finally, it is the target organ of human specific infectious agents, such as Hepatitis C virus. Thus, there is a major interest of biopharmaceutical industries, scientists and clinicians for having human hepatocytes on demand. Studies on animal models can be misleading because the activity, specificity and intermediate metabolites produced by liver enzymes are often different from their human counterparts. Hepatocytes isolated from human liver biopsies represent the ideal source, but availability of liver biopsies is limited and hepatocytes cannot be amplified in vitro. Liver organs that are not suitable for organ transplantation are scarce and of poor quality (massive steatosis in general). This project associates the INSERM UMR 1064 unit (Nantes) and the society Biopredic (Rennes). The UMR1064 Center of Research in Transplantation and Immunology, develops a research area focuses on the induction of tolerance in transplantation and cell therapy in the Liver. Biopredic is an internationally recognized company specializing in the distribution of primary human cells and in particular human hepatocytes for research, drug development, pharmacology and toxicology. The LabCom program aims to develop a robust and reliable system capable to mass-produce human hepatocytes. The liver has an extraordinary ability to regenerate after injury and unique feature to regenerate from residual hepatocytes present in the liver. Currently, it is not possible to reconstruct in vitro the complex architecture of the liver organ capable of replicating hepatocytes. We will develop an innovative technology based on liver regeneration properties to produce rapidly and massively human hepatocytes in vivo in the rat liver, which has the ideal size as a laboratory bioreactor (1 billion hepatocytes per liver). We will develop a method for the purification of human hepatocytes from humanized livers. We will build large cell banks of cryopreserved human hepatocytes of different genotypes ready for commercialization. In addition, a second innovation of the developed technology is to provide an animal model with a humanized liver capable of modeling human liver fibrosis/ cirrhosis and respond to vaccination. Biopredic company will enrich their intellectual properties and its technological know-how, allowing it to be innovative and become a leadership in the industrial environment of a strong international competition. INSERM UMR1064 will economically develop its know-how and have a humanized liver model in rats for its research in immunology of transplantation and liver biotherapy.