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The blood-brain-barrier (BBB) prevents that harmful substances in the blood enter the brain, and barrier disruption has been associated with a variety of central-nervous-system diseases. In vitro BBB models enable to recapitulate the BBB behavior in a controlled environment to investigate how the barrier reacts to stress events and external insults. Here, we present a human-cell-based BBB platform with integrated, transparent electrodes to monitor barrier tightness in real time at high spatiotemporal resolution. The BBB model includes human cerebral endothelial cells and primary human pericytes and astrocytes in a three-dimensional arrangement within a pump-free, open microfluidic platform. With our platform, we demonstrate that oxygen-glucose deprivation (OGD), which mimics the characteristics of an ischemic insult, induces a rapid remodeling of the cellular actin structures and subsequent morphological changes in the endothelial cells. High-resolution live imaging showed the formation of large actin stress-fiber bundles in the endothelial layer during OGD application, which ultimately led to cell shrinkage and barrier breakage. Simultaneous electrical measurements showed a rapid decrease of the barrier electrical resistance before the appearance of the stress fibers, which indicates that the barrier function is compromised already before the appearance of drastic morphological changes. The results demonstrate that our BBB platform recapitulates the main barrier functions in vitro and can be used to investigate the rapid reorganization of the BBB upon application of external stimuli.

Electrophysiological research on neural networks and their activity focuses on the recording and analysis of large data sets that include information of thousands of neurons. CMOS microelectrode arrays (MEAs) feature thousands of electrodes at a spatial resolution on the scale of single cells and are, therefore, ideal tools to support neural-network research. Moreover, they offer high spatio-temporal resolution and signal-to-noise ratio (SNR) to capture all features and subcellular-resolution details of neuronal signaling. Here, we present a dual-mode (DM) MEA, which enables simultaneous: 1) full-frame readout from all electrodes and 2) high-SNR readout from an arbitrarily selectable subset of electrodes. The DM-MEA includes 19 584 electrodes, 19 584 full-frame recording channels with noise levels of 10.4 $\mu \text{V}~_{\text {rms}}$ in the action potential (AP) frequency band (300 Hz–5 kHz), 246 low-noise recording channels with noise levels of 3.0 $\mu \text{V}~_{\text {rms}}$ in the AP band and eight stimulation units. The capacity to simultaneously perform full-frame and high-SNR recordings endows the presented DM-MEA with great flexibility for various applications in neuroscience and pharmacology.

To enable accurate, high-throughput and longer-term studies of the immunopathogenesis of type 1 diabetes (T1D), we established three in-vitro islet-immune injury models by culturing spheroids derived from primary human islets with proinflammatory cytokines, activated peripheral blood mononuclear cells or HLA-A2-restricted preproinsulin-specific cytotoxic T lymphocytes. In all models, β-cell function declined as manifested by increased basal and decreased glucose-stimulated insulin release (GSIS), and decreased intracellular insulin content. Additional hallmarks of T1D progression such as loss of the first-phase insulin response (FFIR), increased proinsulin-to-insulin ratios, HLA-class I expression, and inflammatory cytokine release were also observed. Using these models, we show that liraglutide, a glucagon-like peptide 1 receptor agonist, prevented loss of GSIS under T1D-relevant stress, by preserving the FFIR and decreasing immune cell infiltration and cytokine secretion. Our results corroborate that liraglutide mediates an anti-inflammatory effect that aids in protecting β-cells from the immune-mediated attack that leads to T1D.

Here, we present a microfluidic platform for establishing continuous perfusion in transwell-based in-vitro barrier models. We developed a poly(methylmethacrylate) (PMMA)-based microfluidic device, which allows for dynamic control of culture microenvironments in both, apical and basolateral compartments of cell-covered transwell inserts. We demonstrate the use of the device for high-resolution confocal imaging of the barrier models.

Objective: Techniques to identify monosynaptic connections between neurons have been vital for neuroscience research, facilitating important advancements concerning network topology, synaptic plasticity, and synaptic integration, among others. Approach: Here, we introduce a novel approach to identify and monitor monosynaptic connections using high-resolution dendritic spine Ca2+ imaging combined with simultaneous large-scale recording of extracellular electrical activity by means of high-density microelectrode arrays. Main results: We introduce an easily adoptable analysis pipeline that associates the imaged spine with its presynaptic unit and test it on in vitro recordings. The method is further validated and optimized by simulating synaptically-evoked spine Ca2+ transients based on measured spike trains in order to obtain simulated ground-truth connections. Significance: The proposed approach offers unique advantages as (a) it can be used to identify monosynaptic connections with an accurate localization of the synapse within the dendritic tree, (b) it provides precise information of presynaptic spiking, and (c) postsynaptic spine Ca2+ signals and, finally, (d) the non-invasive nature of the proposed method allows for long-term measurements. The analysis toolkit together with the rich data sets that were acquired are made publicly available for further exploration by the research community., Journal of Neural Engineering, 19 (4), ISSN:1741-2560, ISSN:1741-2552

The selective permeability of the blood-brain barrier (BBB) is a major obstacle to delivery of drugs to the central nervous system (CNS), so that in vitro BBB models are needed for drug permeability studies. Here, we present a pump-free, hanging-drop-based microfluidic BBB model that recapitulates key BBB structural features and functions. BBB function was confirmed by immunofluorescence staining, which showed a high expression of tight-junction proteins, and by its low permeability to the paracellular diffusion of small molecules. The model included a 3D network of pericytes and astrocytes embedded in a hydrogel layer in close proximity to the endothelial cells.

Citizens’ assemblies have gained popularity as instruments of effective and meaningful citizen participation. In the complex transnational context of EU policy-making, citizens’ assemblies can bring citizens and policy-makers closer, promote truly transnational political debates and improve the quality of the EU democracy and policies. But how can citizens’ assemblies be introduced into the EU legal and institutional framework, and its policy processes? What functions should citizens’ assemblies perform? And how should their work be organised to ensure they are democratic, well-functioning and effective instruments of policy-making and citizen participation in the EU? This paper provides answers to these questions by introducing a model for the institutionalisation of European Citizens’ Assemblies. It describes the processes, main bodies and institutions that need to be involved and explores how citizens’ assemblies can be integrated into the EU’s institutional and legal set-up.The proposed model has several distinct features. First and foremost, it is citizen-centred. EU institutions put forward their ideas, but it is randomly selected citizens that steer the process. As members of a Citizens’ Board, they define the agenda for a Citizens’ Assembly by selecting topics and questions, organise deliberations and monitor the implementation of results. Second, the model describes how to connect European Citizens’ Assemblies directly to the EU’s policy-making process. It proposes an Interinstitutional Agreement on deliberative law-making between the EU main institutions as the legal foundation of European Citizens’ Assemblies. It also illustrates how the deliberative cycle of European Citizens’ Assemblies can be effectively connected to the EU policy cycle.The model for European Citizens’ Assemblies is a concrete response to the lessons from the Conference on the Future of Europe. It draws on the experience of the Conference’s European Citizens’ Panels and suggests a format that would make this experience permanent and more impactful. The model presents a logical next step forward—a step that is necessary to move onto the next level of EU citizen participation.

Chronic imaging of neuronal networks in vitro has provided fundamental insights into mechanisms underlying neuronal function. Current labeling and optical imaging methods, however, cannot be used for continuous and long-term recordings of the dynamics and evolution of neuronal networks, as fluorescent indicators can cause phototoxicity. Here, we introduce a versatile platform for label-free, comprehensive and detailed electrophysiological live-cell imaging of various neurogenic cells and tissues over extended time scales. We report on a dual-mode high-density microelectrode array, which can simultaneously record in (i) full-frame mode with 19,584 recording sites and (ii) high-signal-to-noise mode with 246 channels. We set out to demonstrate the capabilities of this platform with recordings from primary and iPSC-derived neuronal cultures and tissue preparations over several weeks, providing detailed morpho-electrical phenotypic parameters at subcellular, cellular and network level. Moreover, we develop reliable analysis tools, which drastically increase the throughput to infer axonal morphology and conduction speed., Nature Communications, 11 (1), ISSN:2041-1723

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.