Advancing tools for predictive toxicology
Award Winners Series: Advancing tools for predictive toxicology
Mar 20, 2019 9:00 AM EDT
Efficient functional cyst formation of biliary epithelial cells using microwells for potential bile duct organisation in vitro
Astia Rizki-Safitri1, Marie Shinohara1, Yasushi Miura2,5, Mathieu Danoy3,4, Minoru Tanaka5,6, Atsushi Miyajima5, & Yasuyuki Sakai1,7,8
1Center for International Research on Integrative Biomedical Systems (CIBiS), Institute of Industrial Science, The University of Tokyo, Japan
2Department of Life Science and Medical Bio-‐Science, School of Advanced Science and Engineering, Waseda University, Japan
3LIMMS/CNRS UMI2820, IIS The University of Tokyo, Japan
4Institut d'Electronique, de Microélectronique et de Nanotechnologies (IEMN), Université Lille, Lille, France
5Laboratory of Cell Growth and Differentiation, Institute of Molecular and Cellular Bioscience, The University of Tokyo, Japan,
6National center for global medicine (NCGM), Tokyo, Japan
7Department of Chemical System Engineering, graduate school of Engineering, The University of Tokyo, Japan
8Max Planck-‐The University of Tokyo, Center for Integrative Inflammology, The University of Tokyo, Japan.
Establishing a bile duct in vitro is valuable to obtain relevant hepatic tissue culture systems for cell-‐ based assays in chemical and drug metabolism analyses. The cyst constitutes the initial morphogenesis for bile duct formation from biliary epithelial cells (BECs) and serves the main building block of bile duct network morphogenesis from the ductal plate during embryogenesis in rodents. Cysts have been commonly cultured via Matrigel-‐embedded culture, which does not allow structural organisation and restricts the productivity and homogeneity of cysts. In this study, we propose a new method utilising oxygen permeable honeycomb microwells for efficient cyst establishment. Primary mouse BECs were seeded on four sizes of honeycomb microwell (46, 76, 126, and 326 μm-‐size in diameter). Matrigel in various concentrations was added to assist in cyst formation. The dimension accommodated by microwells was shown to play an important role in effective cyst formation. Cytological morphology, bile acid transportation, and gene expression of the cysts confirmed the favourable basic bile duct function compared to that obtained using Matrigel-‐embedded culture. Our method is expected to contribute to engineered in vitro liver tissue formation for cell-‐based assays.
SCIENTIFIC Reports | (2018) 8:11086
Generation of recombinant human anti-diphtheria toxin neutralizing antibody to replace equine sera
Esther Wenzel1, Paul Stickings2, Jeffrey Brown3, Thea Sesardic2, Androulla Efstratiou4, Michael Hust1
1Technische Universität Braunschweig, Department of Biotechnology, Braunschweig, Germany
2National Institute for Biological Standards and Control (NIBSC)Division of Bacteriology, Potters Bar, United Kingdom
3PISC, The PETA International Science Consortium Ltd, London, United Kingdom
4Public Health England, London, United Kingdom
Diphtheria is a disease caused by toxigenic Corynebacterium spp. that produce diphtheria toxin (DT). Diphtheria is a significant health problem in countries with poor immunization coverage or disrupted immunization programs. Even in countries where the disease is well controlled, there is a need to maintain a stockpile of therapeutic diphtheria antitoxin (DAT) for management of sporadic or imported cases. Currently, diphtheria is still treated with equine sera in the same way it was treated more than 100 years ago by Emil von Behring. Nowadays, two major strategies are used for the generation of human antibodies: transgenic mice and in vitro selection technologies. Transgenic mice allow the generation of human antibodies using hybridoma technology. The alternative is the generation of human antibodies by antibody phage display which replaces animal immunizations and is based on an in vitro selection process. The aim of the DATMAB project is the generation of neutralizing fully human monoclonal antibodies (mAb) against DT. The long-term goal is the replacement of equine DAT sera with a stockpiled recombinant antibody product produced in cell culture. In the DATMAB project, human antibody fragments (scFv) were generated by phage display against DT using naïve and immune antibody gene libraries. The antibody generation and development follow the 3Rs rules to replace, reduce and refine animal experiments. An MTT-based neutralization assay was used to quantitatively measure metabolic activity of Vero cells and confirm cytotoxic effects due to the presence of DT. Over 650 mAbs were selected by phage display. Over 500 mAbs were sub-cloned and produced as scFv-Fc and 290 scFv-Fc demonstrated significant toxin neutralization activity using a Vero cellbased in vitro assay. Some promising candidates were found and produced as IgG. These highly neutralizing IgGs interact mainly with the receptor binding domain of DT and have a neutralizing potency of up to 445 IU/mg. They will be further characterized regarding stability and long-term storage. Furthermore, combinations of antibodies against different domains will be tested to develop an oligoclonal cocktail of neutralizing antibodies mimicking the mode of action of the currently used serum.
Development and Use of Adverse Outcome Pathway (AOP) Networks to Support Assessment of Organ Level Effects
Marie Scklodowska-Curie Fellow, Liverpool John Moores University, UK.
It is acknowledged that individual Adverse Outcome Pathways (AOPs) trivialise complex real world biological and toxicological scenarios. As a result, there is growing interest to develop networks of AOPs due to their ability to evaluate the interactions along a pathway. Therefore, network modelling relying on both data mapping and analytical analysis offers a fundamental and comprehensive understanding of the mechanistic toxicology. Within this presentation, the concept of AOP network derivation will be introduced and the results of topological analysis will be shown. This exercise allowed for the scoping of relevant AOPs, which served as a starting point for the development of organ specific networks of AOPs and identification of critical paths to extend quantitative organ-to-population-level models for risk assessment. These advances can also be applied to prioritise development of assays, in vitro testing and omics analysis.
Acknowledgement: funding from the EU in3 Marie Skłodowska-Curie Action - Innovative Training Network under grant no. 72197