Project 060

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

07/2016-12/2017

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.

The figure shows the schematic setting of the human in-vitro blood-brain barrier models. The human induced pluripotent stem cells are differentiated into brain endothelial cells (BE ), neural stem cells (NS), astrocytes (AST) and pericytes (PER). HiPSC-derived brain endothelial cells grown on semipermeable membranes form a tight barrier on Transwell inserts. This process is supported by neural stem cells, astrocytes and pericytes, which are situated in the basolateral (CNS mimicking) compartment. In the dynamic, flow-based hollow-fibre model brain endothelial cells are seeded in the lumen of plastic capillaries consisting of semipermeable membranes, whereas neural stem cells, astrocytes and pericytes grow in the extracapillary space (ECS) and secrete factors essential for barrier tightening.

Publications:

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.

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.

Institution

AIT Austrian Institute of Technology GmbH
Department Health & Environment, Molecular Diagnostics
Wien, Österreich

Cooperation Partner

Lehrstuhl Tissue Engineering und Regenerative Medizin (TERM)
Universitätsklinik Würzburg, Deutschland

Duration

07/2016 - 10/2018