Optimisation of a future standard in-vitro model of the human blood-brain barrier
PD Dipl.-Ing. Dr. Winfried Neuhaus & Dr. Marco Metzger AIT Austrian Institute of Technology GmbH, Wien & Lehrstuhl Tissue Engineering und Regenerative Medizin (TERM), Universitätsklinik Würzburg
Currently, no ultimate human BBB in-vitro model exists. Models based on primary cells as well as on immortalized cell lines form barriers with insufficient tightness properties.
The blood-brain barrier (BBB) regulates the transport of substances between blood circulation and the central nervous system (CNS). The BBB can be understood as a kind of bidirectional filter system that is responsible for maintenance of the homeostasis in the CNS. Moreover, the BBB has to protect the CNS against viral and bacterial infections. In several diseases (stroke, brain tumour, Alzheimer’s disease, epilepsy, multiple sclerosis,...) alterations of the BBB‘s functionality could be found, and restoration of the BBB might result in milder disease progressions. In addition, the BBB plays a pivotal role in drug research as well as drug development. Several CNS-targeted compounds can not reach the CNS, because they are recognized by the BBB and effluxed back into the blood-stream. On the other hand, many drugs that act in the periphery should not cross the BBB in order to prevent CNS-adverse side-effects.
Currently, no ultimate human BBB in-vitro model exists. Models based on primary cells as well as immortalized cell lines form barriers with insufficient tightness properties. Cells of the neurovascular unit (NVU) such as astrocytes, pericytes and neural stem cells are known to be capable of inducing BBB properties in brain endothelial cells in vitro. In addition, shear stress mediated by blood flow is able to induce further BBB properties in brain endothelial cells. In the recent time some alternative models have been proposed and introduced which are derived from different stem cell sources.
The aim of the current project was to optimize BBB in vitro models which were in particular based on human induced pluripotent stem cell lines (hiPSC) and include the influence of the microenvironment. During the project protocols were successfully implemented to differentiate brain endothelial cells (BEC), astrocytes (AST), pericytes (PER) and neural stem cells (NSC). These cells were used to develop static Transwell as well as dynamic flow models which were compared to models based on standard immortalized brain endothelial cells (see figure). The comprehensive characterization of the models was accomplished at the functional as well as the molecular level (see Appelt-Menzel et al., Stem Cell Reports, 2017). Cells of the NVU improved barrier properties significantly on the one side, but were also essential to enhance barrier damage in the stroke models on the other side. The established dynamic flow reactors were applicable for chronic long-term experiments over several weeks. The possibilities of applications of these models are very far-ranging in several research areas as well as in drug development processes, and have the potential to reduce and replace – according to the 3Rs-principle – the usage of animal models.
W. Neuhaus (2017) “Human induced pluripotent stem cell (hiPSC) based in vitro models of the blood-brain barrier: The future standard ?” Neural Regeneration Research 12(10):1607-1609.
Master thesis Anna Sophia Wilhelm (2017) „Optimization of a human blood-brain barrier in vitro model - Investigation of the influence of dynamic flow-culture conditions and implementation of non-invasive impedance spectroscopy“, Julius-Maximilians-Universität Würzburg.
Master thesis Elsa Görsch (2017) „Optimization of Human Blood-Brain Barrier Models“, Julius- Maximilians-Universität Würzburg.
A. Appelt-Menzel, A. Cubukova, K. Gunther, F. Edenhofer, J. Piontek, G. Krause, T. Stuber, H. Walles, W. Neuhaus and M. Metzger (2017). "Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells." Stem Cell Reports 8(4): 894-906.
A. Appelt-Menzel, A. Cubukova and M. Metzger (2018). “Establishment of a human bloodbrain barrier co-culture model mimicking the neurovascular unit using induced pluripotent stem cells.” Current Protocols in Stem Cell Biology, 47, e62.
A. Ramme, L. Koenig, C. Schwenk, C. Magauer, D. Faust, A. Lorenz, A. Krebs, C. Drewell, K. Schirrmann, A. Vladetic, G. Lin, S. Pabinger, W. Neuhaus, F. Bois, R. Lauster, U. Marx, E. Dehne (2018) „Towards an autologous iPSC-derived patient-on-a-chip"; bioRxiv, 1, S. 1-26.
A. Ramme, L. Koenig, T. Hasenberg, C. Schwenk, C. Magauer, D. Faust, A. Lorenz, A. Krebs, C. Drewell, K. Schirrmann, A. Vladetic, G. Lin, S. Pabinger, W. Neuhaus, F. Bois, R. Lauser, U. Marx, E. Dehne (2019): "Autologous iPSC-derived four-organ-chip"; Future Science OA, 8, 5; S. 1 - 12.
Master thesis Nadja Pracser (2019) “The role of the microenvironment in a human blood-brain barrier in vitro model of ischemia"; University of Vienna.
A Gerhartl, N Pracser, A Vladetic, S Hendrikx, HP Friedl & W Neuhaus (2020). The pivotal role of micro-environmental cells in a human blood–brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis. Fluids Barriers CNS 17, 19.
A. Appelt-Menzel, S. Oerter, S. Mathew, U. Haferkamp, C. Hartmann, M. Jung, W. Neuhaus, O. Pless (2020). Human iPSC-Derived Blood-Brain Barrier Models: Valuable Tools for Preclinical Drug Discovery and Development? Curr Protoc Stem Cell Biol; 55(1):e122. doi: 10.1002/cpsc.122.
AIT Austrian Institute of Technology GmbH Department Health & Environment, Molecular Diagnostics Wien, Österreich
Lehrstuhl Tissue Engineering und Regenerative Medizin (TERM) Universitätsklinik Würzburg, Deutschland
Studies of Food- and Biotechnology at the University of Natural Resources and Life Sciences in Vienna. Doctoral thesis on the development and validation of blood-brain barrier in-vitro models. Assistant and lecturer at the technical college "Biotechnology". Group leader of the Preclinical and Blood-Brain Barrier (BBB) Research at Pharmacon. 2010-2016 research at the University Hospital in Würzburg, as well as at the Department of Pharmaceutical Chemistry and the Institute of medical Genetics in Vienna. Since 2016 research group leader at AIT.
Dr. Marco Metzger
Studies of Biotechnology at the University of Applied Sciences Mannheim, dissertation at the University Tübingen on “Influence of the Repulsive Guidance Molecule (RGM) on proliferation and differentiation on intestinal stem cells”. Post-doc at the Institute of Childs Health of the University College London (UK). 2009-2011 own research group at the Translational Centre for Regenerative Medicine at University of Leipzig. 2011 group leader at the Fraunhofer IGB project group “Oncology” and the Chair for Tissue Engineering & Regenerative Medicine (TERM) at the University Hospital Würzburg. Since 2014 head of the “Implants” unit at the Translational Centre Würzburg.
Anna Gerhartl, MSc.
Bachelor in Oxford. Master studies in Medical and Pharmaceutical Biotechnology at the University of Applied Sciences Krems, thesis on „Patient-derived iPSCs as a Tool for Modeling Cortical Migration Defects”. Now PhD student in the group of Winfried Neuhaus.
Dipl.-Ing. (FH) Antje Appelt-Menzel
Engineering Studies of Pharmaceutical Biotechnology in Jena. Diploma thesis on “Optimization of isolation and culturing conditions of human keratinocytes and fibroblasts and construction of a 3D skin model for automated production” in 2009 at the Fraunhofer IGB in Stuttgart. 2009-2012 engineer at the Chair Tissue Engineering and Regenerative Medicine of the University Hospital Würzburg. Dissertation on “Establishment and qualification of a human blood-brain barrier model based on human induced pluripotent and multipotent stem cells”. Now Post-doc in the team of Marco Metzger.