Microelectrode Arrays Measure Blocking of Voltage-Gated Calcium Ion Channels on Supported Lipid Bilayers Derived from Primary Neurons.

Drug studies targeting neuronal ion channels are crucial to understand neuronal function and develop therapies for neurological diseases. The traditional method to study neuronal ion-channel activities heavily relies on the whole-cell patch clamp as the industry standard. However, this technique is both technically challenging and labour-intensive, while involving the complexity of keeping cells alive with low throughput. Therefore, the shortcomings are limiting the efficiency of ion-channel-related neuroscience research and drug testing. Here, this work reports a new system of integrating neuron membranes with organic microelectrode arrays (OMEAs) for ion-channel-related drug studies. This work demonstrates that the supported lipid bilayers (SLBs) derived from both neuron-like (neuroblastoma) cells and primary neurons are integrated with OMEAs for the first time. The increased expression of voltage-gated calcium (CaV) ion channels on differentiated SH-SY5Y SLBs  compared to non-differentiated ones is sensed electrically. Also, dose-response of the CaV ion-channel blocking effect on primary cortical neuronal SLBs from rats is monitored. The dose range causing ion channel blocking is comparable to literature. This system overcomes the major challenges from traditional methods (e.g., patch clamp) and showcases an easy-to-test, rapid, ultra-sensitive, cell-free, and high-throughput platform to monitor dose-dependent ion-channel blocking effects on native neuronal membranes.

3D organic bioelectronics for electrical monitoring of human adult stem cells.

Three-dimensional in vitro stem cell models have enabled a fundamental understanding of cues that direct stem cell fate. While sophisticated 3D tissues can be generated, technology that can accurately monitor these complex models in a high-throughput and non-invasive manner is not well adapted. Here we show the development of 3D bioelectronic devices based on the electroactive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate)-(PEDOT:PSS) and their use for non-invasive, electrical monitoring of stem cell growth. We show that the electrical, mechanical and wetting properties as well as the pore size/architecture of 3D PEDOT:PSS scaffolds can be fine-tuned simply by changing the processing crosslinker additive. We present a comprehensive characterization of both 2D PEDOT:PSS thin films of controlled thicknesses, and 3D porous PEDOT:PSS structures made by the freeze-drying technique. By slicing the bulky scaffolds we generate homogeneous, porous 250 µm thick PEDOT:PSS slices, constituting biocompatible 3D constructs able to support stem cell cultures. These multifunctional slices are attached on indium-tin oxide substrates (ITO) with the help of an electrically active adhesion layer, enabling 3D bioelectronic devices with a characteristic and reproducible, frequency dependent impedance response. This response changes drastically when human adipose derived stem cells (hADSCs) grow within the porous PEDOT:PSS network as revealed by fluorescence microscopy. The increase of cell population within the PEDOT:PSS porous network impedes the charge flow at the interface between PEDOT:PSS and ITO, enabling the interface resistance (R1) to be used as a figure of merit to monitor the proliferation of stem cells. The non-invasive monitoring of stem cell growth allows for the subsequent differentiation 3D stem cell cultures into neuron like cells, as verified by immunofluorescence and RT-qPCR measurements. The strategy of controlling important properties of 3D PEDOT:PSS structures simply by altering processing parameters can be applied for development of a number of stem cell in vitro models as well as stem cell differentiation pathways. We believe the results presented here will advance 3D bioelectronic technology for both fundamental understanding of in vitro stem cell cultures as well as the development of personalized therapies.

A human-derived neurovascular unit in vitro model to study the effects of cellular cross-talk and soluble factors on barrier integrity.

The blood-brain barrier (BBB) restricts paracellular and transcellular diffusion of compounds and is part of a dynamic multicellular structure known as the "neurovascular unit" (NVU), which strictly regulates the brain homeostasis and microenvironment. Several neuropathological conditions (e.g., Parkinson's disease and Alzheimer's disease), are associated with BBB impairment yet the exact underlying pathophysiological mechanisms remain unclear. In total, 90% of drugs that pass animal testing fail human clinical trials, in part due to inter-species discrepancies. Thus, in vitro human-based models of the NVU are essential to better understand BBB mechanisms; connecting its dysfunction to neuropathological conditions for more effective and improved therapeutic treatments. Herein, we developed a biomimetic tri-culture NVU in vitro model consisting of 3 human-derived cell lines: human cerebral micro-vascular endothelial cells (hCMEC/D3), human 1321N1 (astrocyte) cells, and human SH-SY5Y neuroblastoma cells. The cells were grown in Transwell hanging inserts in a variety of configurations and the optimal setup was found to be the comprehensive tri-culture model, where endothelial cells express typical markers of the BBB and contribute to enhancing neural cell viability and neurite outgrowth. The tri-culture configuration was found to exhibit the highest transendothelial electrical resistance (TEER), suggesting that the cross-talk between astrocytes and neurons provides an important contribution to barrier integrity. Lastly, the model was validated upon exposure to several soluble factors [e.g., Lipopolysaccharides (LPS), sodium butyrate (NaB), and retinoic acid (RA)] known to affect BBB permeability and integrity. This in vitro biological model can be considered as a highly biomimetic recapitulation of the human NVU aiming to unravel brain pathophysiology mechanisms as well as improve testing and delivery of therapeutics.

Organic electronic transmembrane device for hosting and monitoring 3D cell cultures.

3D cell models have made strides in the past decades in response to failures of 2D cultures to translate targets during the drug discovery process. Here, we report on a novel multiwell plate bioelectronic platform, namely, the e-transmembrane, capable of supporting and monitoring complex 3D cell architectures. Scaffolds made of PEDOT:PSS [poly(3,4-ethylenedioxythiophene):polystyrene sulfonate] are microengineered to function as separating membranes for compartmentalized cell cultures, as well as electronic components for real-time in situ recordings of cell growth and function. Owing to the high surface area-to-volume ratio, the e-transmembrane allows generation of deep, stratified tissues within the porous bulk and cell polarization at the apico-basal domains. Impedance spectroscopy measurements carried out throughout the tissue growth identified signatures from different cellular systems and allowed extraction of critical functional parameters. This platform has the potential to become a universal tool for biologists for the next generation of high-throughput drug screening assays.

Conducting Polymer-ECM Scaffolds for Human Neuronal Cell Differentiation.

3D cell culture formats more closely resemble tissue architecture complexity than 2D systems, which are lacking most of the cell-cell and cell-microenvironment interactions of the in vivo milieu. Scaffold-based systems integrating natural biomaterials are extensively employed in tissue engineering to improve cell survival and outgrowth, by providing the chemical and physical cues of the natural extracellular matrix (ECM). Using the freeze-drying technique, porous 3D composite scaffolds consisting of poly(3,4-ethylene-dioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS), containing ECM components (i.e., collagen, hyaluronic acid, and laminin) are engineered for hosting neuronal cells. The resulting scaffolds exhibit a highly porous microstructure and good conductivity, determined by scanning electron microscopy and electrochemical impedance spectroscopy, respectively. These supports boast excellent mechanical stability and water uptake capacity, making them ideal candidates for cell infiltration. SH-SY5Y human neuroblastoma cells show enhanced cell survival and proliferation in the presence of ECM compared to PEDOT:PSS alone. Whole-cell patch-clamp recordings acquired from differentiated SHSY5Y cells in the scaffolds demonstrate that ECM constituents promote neuronal differentiation in situ. These findings reinforce the usability of 3D conducting supports as engineered highly biomimetic and functional in vitro tissue-like platforms for drug or disease modeling.

Investigation of Host-Microbe-Parasite Interactions in an In Vitro 3D Model of the Vertebrate Gut.

In vitro models of the gut-microbiome axis are in high demand. Conventionally, intestinal monolayers grown on Transwell setups are used to test the effects of commensals/pathogens on the barrier integrity, both under homeostatic and pathophysiological conditions. While such models remain valuable for deepening the understanding of host-microbe interactions, often, they lack key biological components that mediate this intricate crosstalk. Here, a 3D in vitro model of the vertebrate intestinal epithelium, interfaced with immune cells surviving in culture for over 3 weeks, is developed and applied to proof-of-concept studies of host-microbe interactions. More specifically, the establishment of stable host-microbe cocultures is described and functional and morphological changes in the intestinal barrier induced by the presence of commensal bacteria are shown. Finally, evidence is provided that the 3D vertebrate gut models can be used as platforms to test host-microbe-parasite interactions. Exposure of gut-immune-bacteria cocultures to helminth "excretory/secretory products" induces in vivo-like up-/down-regulation of certain cytokines. These findings support the robustness of the modular in vitro cell systems for investigating the dynamics of host-microbe crosstalk and pave the way toward new approaches for systems biology studies of pathogens that cannot be maintained in vitro, including parasitic helminths.

Functional Infectious Nanoparticle Detector: Finding Viruses by Detecting Their Host Entry Functions Using Organic Bioelectronic Devices.

Emerging viruses will continue to be a threat to human health and wellbeing into the foreseeable future. The COVID-19 pandemic revealed the necessity for rapid viral sensing and inhibitor screening in mitigating viral spread and impact. Here, we present a platform that uses a label-free electronic readout as well as a dual capability of optical (fluorescence) readout to sense the ability of a virus to bind and fuse with a host cell membrane, thereby sensing viral entry. This approach introduces a hitherto unseen level of specificity by distinguishing fusion-competent viruses from fusion-incompetent viruses. The ability to discern between competent and incompetent viruses means that this device could also be used for applications beyond detection, such as screening antiviral compounds for their ability to block virus entry mechanisms. Using optical means, we first demonstrate the ability to recapitulate the entry processes of influenza virus using a biomembrane containing the viral receptor that has been functionalized on a transparent organic bioelectronic device. Next, we detect virus membrane fusion, using the same, label-free devices. Using both reconstituted and native cell membranes as materials to functionalize organic bioelectronic devices, configured as electrodes and transistors, we measure changes in membrane properties when virus fusion is triggered by a pH drop, inducing hemagglutinin to undergo a conformational change that leads to membrane fusion.

Synthesis and characterisation of biocompatible organic-inorganic core-shell nanocomposite particles based on ureasils.

Organic-inorganic core-shell nanocomposites have attracted increasing attention for applications in imaging, controlled release, biomedical scaffolds and self-healing materials. While tunable properties can readily be achieved through the selection of complementary building blocks, synergistic enhancement requires management of the core-shell interface. In this work, we report a one-pot method to fabricate hybrid core-shell nanocomposite particles (CSNPs) based on ureasils. The native structure of ureasils, which are poly(oxyalkylene)/siloxane hybrids, affords formation of an organic polymer core via nanoprecipitation, while the terminal siloxane groups act as a template for nucleation and growth of the silica shell via the Stöber process. Through optimisation of the reaction conditions, we demonstrate the reproducible synthesis of ureasil CSNPs, with a hydrodynamic diameter of ∼150 nm and polydispersity <0.2, which remain electrostatically stabilised in aqueous media for >50 days. Selective functionalisation, either through the physical entrapment of polarity-sensitive fluorescent probes (coumarin 153, pyrene) or covalent-grafting to the silica shell (fluorescein isothiocyanate) is also demonstrated and provides insight into the internal environment of the particles. Moreover, preliminary studies using a live/dead cell assay indicate that ureasil CSNPs do not display cytotoxicity. Given the simple fabrication method and the structural tunability and biocompatability of the ureasils, this approach presents an efficient route to multifunctional core-shell nanocomposite particles whose properties may be tailored for a targeted application.