Archived Webinar

Advancing tools for predictive toxicology

Title: Establishment of bile duct tubular structure mimicking the intrahepatic bile duct morphogenesis for an in vitro bile recovery
Presenters: Astia Rizki-­‐Safitri, Marie Shinohara, Minoru Tanaka, and Yasuyuki Sakai

Title: Generation of recombinant human anti-diphtheria toxin neutralizing antibody to replace equine sera
Presenters: Esther Wenzel, Paul Stickings, Jeffrey Brown, Thea Sesardic, Androulla Efstratiou, Michael Hust

Title: Development and Use of Adverse Outcome Pathway (AOP) Networks to Support Assessment of Organ Level Effects
Presenters: Nicoleta Spinu, Mark TD Cronin, Steven J. Enoch, and Judith C Madden

Award Winners Series: Advancing tools for predictive toxicology
Mar 20, 2019 9:00 AM EDT

Establishment of bile duct tubular structure mimicking the intrahepatic bile duct morphogenesis for an in vitro bile recovery

Astia Rizki-­‐Safitri1,2, Marie Shinohara2,3, Minoru Tanaka4,5, and Yasuyuki Sakai1,2,3,6

1Department of Bioengineering, Graduate School of Engineering, University of Tokyo, Japan
2Center for International Research on Integrative Biomedical Systems (CIBiS), Institute of Industrial Science (IIS), University of Tokyo, Japan
3Department of Chemical System Engineering, Graduate School of Engineering, University of Tokyo, Japan
4Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences (IQB), University of Tokyo, Japan
5National center for global medicine (NCGM), Tokyo, Japan
6Max Planck-­‐The University of Tokyo, Center for Integrative Inflammology, University of Tokyo, Japan Abstract

Bile ducts accumulate and transport metabolite-­‐rich bile necessary for chemical and drug metabolism in the liver. These structures rely on complex differentiation signaling, including the activation of some major gene expressions i.e.: Notch2 and Jagged1. In the in vitro liver model, recent bile duct-­‐like structure shows less morphology resemblance to the in vivo bile duct and less applicable for in vitro bile recovery owing to extracellular matrix (ECM)-­‐embedded culture. We establish an in vitro bile duct tubular structure employing hierarchical liver cell cocultures mimicking the in vivo bile duct morphogenesis, which more closely resembles in vivo bile duct structure and shows metabolite accumulation from hepatocytes. A primary rat hepatoblast was cocultured with biliary epithelial cell (BEC) and mouse embryonic fibroblast (MEF) overlaid by ECM containing Matrigel and collagen gel mixture. A bile duct tubular structure was developed abundantly in a triple cell-­‐coculture system and was able to accumulate a bile analogue from the hepatocyte bile canaliculi. This study demonstrates a development of in vitro bile duct tubular structure as well as the possibility for in vitro bile recovery, so that may contribute to the improvement of liver model and in vitro chemical-­‐drug testing.


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

Nicoleta Spinu1, Mark TD Cronin2, Steven J. Enoch2, and Judith C Madden2

1Marie Scklodowska-Curie Fellow, Liverpool John Moores University, UK; 2Liverpool 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