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Development and optimisation of bioelectronic monitoring techniques like microelectrode array-based field potential measurement and impedance spectroscopy for the functional, label-free and non-invasive monitoring of in vitro neuronal networks is widely investigated in the field of biosensors. Thus, these techniques were individually used to demonstrate the capabilities of, e.g., detecting compound-induced toxicity in neuronal culture models. In contrast, extended application for investigating the effects of central nervous system infecting viruses are rarely described. In this context, we wanted to analyse the effect of herpesviruses on functional neuronal networks. Therefore, we developed a unique hybrid bioelectronic monitoring platform that allows for performing field potential monitoring and impedance spectroscopy on the same microelectrode. In the first step, a neuronal culture model based on primary hippocampal cells from neonatal rats was established with reproducible and stable synchronised electrophysiological network activity after 21 days of cultivation on microelectrode arrays. For a proof of concept, the pseudorabies model virus PrV Kaplan-ΔgG-GFP was applied and the effect on the neuronal networks was monitored by impedance spectroscopy and field potential measurement for 72 h in a multiparametric mode. Analysis of several bioelectronic parameters revealed a virus concentration-dependent degeneration of the neuronal network within 24-48 h, with a significant early change in electrophysiological activity, subsequently leading to a loss of activity and network synchronicity. In conclusion, we successfully developed a microelectrode array-based hybrid bioelectronic measurement platform for quantitative monitoring of pathologic effects of a herpesvirus on electrophysiological active neuronal networks.

Miniaturization and integration of chemical reactions into fluidic systems in combination with product purification or buffer exchange can reduce the amount of solvents and reactants required while increasing synthesis efficiency. A critical step is the regulation of flow rates to realize optimal synthesis conditions and high purification rates, so real-time, label-free monitoring is required in methods such as free-flow electrophoresis. Optical detection methods are widely used, but they often have complex excitation and detection setups that are disadvantageous for point-of-care applications. The method we have chosen is electrochemical impedance spectroscopy for detecting charged compounds in aqueous buffers with low ionic strength. Propranolol was selected for proof of concept and was separated from the organic solvent and the precursor oxirane by free-flow electrophoresis. For this purpose, electrode structures were fabricated in microfluidic channels by photolithographic lift-off technique and optimized in terms of positioning, electrode size and distance for sensitive detection, and quantification of propranolol in the nanomolar range. It is also noteworthy that the organic solvent dimethyl sulfoxide (DMSO) could be detected and quantified by an increased impedance magnitude. Subsequently, the optimized interdigital electrode structures were integrated into the outlet channels of the electrophoretic separation chamber to monitor the various outgoing fluidic streams and provide in-line control of the fluidic flows for the purification step. In conclusion, we can provide a microfluidic chip to monitor the separation efficiency of a substance mixture during free-flow electrophoresis without the need of complex analytical techniques using electrochemical impedance spectroscopy.

Three-dimensional cell models represent the native in vivo situation more closely than two-dimensional cultures and are therefore preferred today for in vitro studies. In this context, there is a great demand for fast, non-invasive, real-time, and label-free methods that are capable for detailed analyses of three-dimensional cultures. To characterize heterogeneous cultures or to detect localized drug effects, a measurement method such as impedance spectroscopy in combination with microcavity arrays (MCAs) is desirable, which additionally offers spatial resolution. To overcome these limitations of the previously described MCA based on opaque silicon substrates and a square shape with four measurement electrodes imposed by the crystal structure, we used the selective laser etching (SLE) method to fabricate microcavities in fused silica and borosilicate glass without geometric constraints. We successfully developed MCAs with variable base including up to eight measurement electrodes in one cavity, which allows the increase in the number of electrode combinations to improve spatial resolution. In addition, we integrated a central cone electrode at the cavity bottom to extend the spatial resolution on the z-axis. To demonstrate the capability of the MCAs, we used MDA-HB-231 spheroids with an enclosed glass sphere to show that the heterogeneity of the model is evident in the relative impedance spectra. Analyses on various cell spheroids highlight the broad applicability of glass MCAs. In conclusion, our SLE-fabricated MCA clearly improve bioelectronic analyses of cellular changes in heterogeneous 3D models. Thus, bioelectronic analysis of electrophysiologically active cells and tumor biopsy samples could significantly benefit from our development., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Microelectrode arrays (MEA) are widely used for bioelectronic monitoring of alterations in cells and tissues. MEAs are based on a substrate that is structured with electrodes and conducting paths. While cheap substrates like printed circuit board materials offers easy production and flexible contacting, there are limitations regarding microstructure resolution, optical transparency and biocompatibility. In contrast, glass substrates are favored due to its biocompatibility, chemical resistance and optical transparency. Drawbacks are high substrate costs and limited flexibility for routing of conducting paths. To overcome these limitations, we wanted to use optical transparent polymer-based substrates. Therefore, we identified the polymer poly-methyl-methacrylate (PMMA) as a promising substrate material, due to its good optical and mechanical properties as well as biocompatibility. To achieve sufficient chemical resistance for high resolution photolithographic structuring a novel process had to be developed involving a protection coating. After optimization of the structuring process, we achieved a comparable resolution and thus, microelectrodes with diameter of less than 100 μm. Moreover, the use of PMMA allowed the simple integration of more than 400 vias directly into the substrate for contacting of the microelectrode array from the bottom without the need of complex and error prone redirecting adapters with hundreds of additional bonding sides. In order to show that the PMMA based MEA is comparable to glass based MEA in terms of signal quality and sensitivity as well as optical and surface properties, we cultivated different cell models on the MEAs and validated our 96-well PMMA MEAs by different bioelectronic monitoring techniques., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Microelectrode arrays (MEAs) are widely used to study the behavior of cells noninvasively and in real time. While the design of MEAs focuses mainly on the electrode material or its application-dependent modification, the passivation layer, which is crucial to define the electrode area and to insulate the conducting paths, remains largely unnoticed. Because often most cells are in direct contact with the passivation layer rather than the electrode material, biocompatible photoresists such as SU-8 are almost exclusively used. However, SU-8 is not without limitations in terms of optical transmission, optimal cell support, or compatibility within polymer-based microfluidic lab on chip systems. Here, we established a silicon nitride (SiN) passivation by physical vapor deposition (PVD), which was optimized and evaluated for impedance spectroscopy-based monitoring of cells. Surface characteristics, biocompatibility, and electrical insulation capability were investigated and compared to SU8 in detail. To investigate the influence of the SiN passivation on the impedimetric analysis of cells, HEK-293 A and MCF-7 were chosen as adherent cell models and measured on microelectrodes of 50-200 μm in diameter. The results clearly revealed an overall suitability of SiN as alternative passivation. While for the smallest electrode size a cell line dependent comparable or slightly decreased cell signal could be observed in comparison with SU-8, a significant higher cell signal was observed for microelectrodes larger than 50 μm in diameter. Furthermore, a high suitability for the bonding of PEGDA and PDMS microfluidic structures on the SiN passivation layer without any leakage could be demonstrated.

In bioelectrocatalysis, immobilised redox enzymes are activated in a bioelectronic interface without redox equivalents such as NADPH, thus enabling heterogeneous flow chemistry. The functional contact between enzyme and electrode requires a high degree of optimisation regarding choice of electrode material, electrode pre-treatment, enzyme immobilisation and reaction conditions. So far, however, there are no systems that can easily enable an optimisation procedure at a higher throughput. Here, we present an advanced platform with a vertical divided cell architecture in conjunction with a developed 96-multipotentiostat to be able to drive redox enzymes in 96 well microtiter plate based multielectrode arrays. This platform controls 96 independent three-electrode setups with arbitrary working electrode materials. We demonstrate its applicability in a mutation study of cytochrome P450 BM3 using indium tin oxide as electrode material and the 7-ethoxycoumarin product quantification assay. We show that the bioelectrocatalytic activity of P450 BM3 can be amplified when the cofactor FAD is erased from the enzyme by a single point mutation, so that FMN becomes the first electron entry point. Bioelectrocatalysis thus offers an approach to enzyme simplification as a remedy for the inherent instability of self-sufficient cytochrome P450 enzymes. In addition, we examined native and artificial enzyme activation with respect to ionic strength and buffer composition. The optimal conditions of the activation types differ substantially from each other and exhibit a new molecular facet in enzyme characteristics. In a proof-of-principle we demonstrate that the platform is also compatible with raw cell extracts, thus opening the door for random mutagenesis screenings.

Insufficient endothelialization of cardiovascular devices is a high-risk factor for implant failure. Presentation of extracellular matrix (ECM)-derived coatings is a well-known strategy to improve implant integration. However, the complexity of the system is challenging and strategies for applying multifunctionality are required. Here, we engineered mussel-derived surface-binding peptides equipped with integrin (c[RGDfK]) and proteoglycan binding sites (FHRRIKA) for enhanced endothelialization. Surface-binding properties of the platform containing l-3,4-dihydroxyphenylalanine (DOPA) residues were confirmed for hydrophilized polycaprolactone-co-lactide scaffolds as well as for glass and polystyrene. Further, heparin and the heparin-binding angiogenic factors VEGF, FGF-2 and CXCL12 were immobilized onto the peptide in a modular assembly. Presentation of bioactive peptides greatly enhanced human umbilical vein endothelial cell (HUVEC) adhesion and survival under static and fluidic conditions. In subsequent investigations, peptide-heparin-complexes loaded with CXCL12 or VEGF had an additional increasing effect on cell viability, differentiation and migration. Finally, hemocompatibility of the coatings was ensured. This study demonstrates that coatings combining adhesion peptides, glycosaminoglycans and modulators are a versatile tool to convey ECM-inspired multifunctionality to biomaterials and efficiently promote their integration.

Understanding of cell migration and spreading out of tumor tissue is of great interest concerning the mechanism and causes of tumor malignancy and metastases. Although there are methods available for studying cell migration on monolayer cell cultures like transwell assays, novel techniques for monitoring cell spreading out of 3D organoids or tumor tissue samples are highly required. In this context, we developed an innovative high-dense microelectrode array for impedimetric monitoring of cell migration from 3D tumor cultures. For a proof of concept, a strongly migrating breast cancer cell line (MDA-MB-231) and two malignant melanoma cell lines (T30.6.9, T12.8.10ZII) were used for generating viable micro-tumor models. The migration propensity was determined by impedimetric monitoring over 144 hours, correlated by microscopy and validated by transwell assays. The impedimetric analysis of covered electrodes and the relative impedance maximum values revealed extended information regarding the contribution of proliferative effects. More strikingly, using reference populations of mitomycin C treated spheroids where proliferation was suppressed, distinction of proliferation and migration was possible. Therefore, our high-dense microelectrode array based impedimetric migration monitoring has the capability for an automated quantitative analysis system that can be easily scaled up as well as integrated in lab on chip devices.

The continuous separation mechanism of micro free-flow electrophoresis (μFFE) is a straightforward, suitable tool for microscale purification of reaction mixtures. However, aqueous separation buffers and organic reaction solvents limit the applicability of this promising combination. Herein, we have explored nonaqueous micro free-flow electrophoresis for this purpose and present its suitability for a continuous workup of organic reactions performed in acetonitrile. After successful nonaqueous FFE separation of organic dyes, the approach was applied to continuously recover the photocatalyst [Ru(bpy) 3 ] 2+ from a homogeneous, acetonitrile-based reaction mixture. This approach opens up possibilities for further downstream processing of purified products and is also attractive for recycling of precious catalyst species.

Large-ring cyclodextrins (CD) are cyclic glucans composed of 9 or more α-1,4-linked glucose units. They are minor side products of bacterial glucanotransferases (CGTases, EC 2.4.1.19) and have previously been available only in very small amounts for studies of their properties in supramolecular complex formation reactions. We engineered a CGTase to synthesize mainly large-ring CD facilitating their preparation in larger amounts. By reversed phase chromatography, we obtained single CD samples composed of 10 to 12 glucose units (CD10, CD11, and CD12) with a purity of >90 %. Their identity was confirmed by high resolution mass spectrometry and fragmentation analysis. We demonstrated the non-toxicity of CD10-CD12 for human cell lines by a cell proliferation assay and impedimetric monitoring. We then showed that CD10 and CD11 are efficient chiral selectors for the capillary electrophoretic separation of the enantiomeric pharmaceuticals fluvastatin, mefloquine, carvedilol, and primaquine., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Multielectrode array (MEA) technology is widely used for the bioelectronic monitoring of cellular alterations. In general, noble metal based MEAs are preferred e.g. for impedance spectroscopy because of their high conductivity and biocompatibility. Today's research focuses on combining different readout methods in a single measurement setup, such as sensitive electronic and optical readouts, where noble metal-based electrodes are excluded and transparent electrodes and optimized MEAs are required. In this context, we used optical transparent indium tin oxide (ITO) as electrode material. As a drawback, the decreased conductivity can lead to drastically decreased cell signals and it is hardly to predict which layout changes lead to a substantial signal increase. To overcome this limitation, we introduce an approach where equivalent circuit modelling (ECM) on reference multielectrode arrays is used to determine cell type specific electrical parameters, which then are used in finite element method (FEM) simulations to predict achievable cell signals and signal-noise-ratios (SNR) and thus use simulation to efficiently optimize multielectrode arrays. To evaluate our approach, MEAs with a wide range of electrode sizes were fabricated with ITO and gold. HEK-A cells were used to compare achievable cell signals for impedimetric monitoring. Our study revealed that especially for large ITO electrodes, the sensitivity drastically decreases. To overcome this drawback, we designed an optimized dual layer ITO MEA with gold support structures and more strikingly, successfully predict the cell signal increase by using our combined ECM and FEM simulation based approach., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Human pluripotent stem cell derived cardiomyocytes are a promising cell source for research and clinical applications like investigation of cardiomyopathies and therefore, identification and testing of novel therapeutics as well as for cell based therapy approaches. However, actually it´s a challenge to generate matured adult cardiomyocyte-like phenotype in a reasonable time. Moreover, there is a lack of applicable non-invasive label-free monitoring techniques providing quantitative parameters for analysing the culture stability and maturation status. In this context, we established an efficient protocol based on a combined differentiation of hiPSC in 2D cultures followed by a forced reaggregation step that leads to highly enriched (>90% cardiomyocytes) cardiomyocyte clusters. Interestingly, 3D cultures revealed an accelerated maturation as well as phenotype switch from atrial to ventricular cardiomyocytes. More strikingly using combined impedimetric and electrophysiological monitoring the high functionality and long-term stability of 3D cardiomyocyte cultures, especially in comparison to 2D cultures could be demonstrated. Additionally, chronotropic as well as QT-prolongation causing reference compounds were used for validating the cardio specific and sensitive reaction over the monitored time range of more than 100 days. Thus, the approach of multiparametric bioelectronic monitoring offers capabilities for the long-term quantitative analysis of hiPS derived cardiomyocyte culture functionality and long-term stability. Moreover, the same multiparametric bioelectronic platform can be used in combination with validated long-term stable cardiomyocyte cultures for the quantitative detection of compound induced effects. This could pave the way for more predictive in vitro chronic/repeated dose cardiotoxicity testing assays., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The maximum response and 10-year survival rate for metastatic melanoma patients treated with standardised chemotherapy is still less than 15% and 10%, respectively. In contrast, oncogene targeting was found a promising tool for killing of BRAF V600 mutated melanoma cells. Nevertheless, despite improved response and survival rates, resistance acquisition remains an ongoing problem. In this context, the impact of chronic BRAF inhibition on the efficacy of commonly applied cytostatics is still unknown. In our study, human melanoma cells with BRAF V600E mutation were treated with chemotherapeutics and a BRAF inhibitor. Resistance patterns were analysed by microelectrode array-based impedance spectroscopy, XTT and flow cytometric apoptosis/proliferation assay. BRAF V600E melanoma cells acquired a time- and concentration-dependent desensitisation up to 100-fold towards oncogene-specific PLX4032 and chemotherapeutic dacarbazine after twelve months treatment. The impact of multiple drug insensitivity on molecular melanoma characteristics was elaborated via mRNA and protein quantification. Following BRAF V600E targeting, melanoma cells developed an increasingly aggressive, dacarbazine-insensitive phenotype. Thereby, hyperactivated canonical alternative MAPK and bypass PI3K/AKT signalling caused cross-resistance of differently acting drugs. With these results, we are the first to show that long-term melanoma therapy with BRAF inhibitors can prevent further therapeutic success with dacarbazine due to acquisition of cross-resistance.

In today's development of anticancer drugs, there is an enormous demand for sensitive, non-invasive real-time screening technologies to identify pharmacodynamics/-kinetics of single and combined drugs with high precision. The combination of sophisticated drug sensitivity testing with advanced in vitro tumor models reflecting heterogeneous tumor behavior in vivo is needed to more reasonably predict therapeutic outcome in vivo. In this study, the benefits of our real-time, non-invasive multidimensional impedance platform over standard in vitro drug sensitivity assays were demonstrated quantitatively using an advanced melanoma model. Detailed pharmacological profiles of clinically established targeted therapeutics in single and combination treatment have been identified in patient tissue and isolated 2D/3D cell line cultures. Impedance spectroscopy revealed significant differences in tissue structure responsible for BRAF inhibitor pharmacokinetics in BRAF V600E tumor microfragments and cell lines. Remarkably, BRAF-/MEK inhibitor combination treatment of direct patient-derived tissue, but not melanoma cell lines, resulted in short-term antagonistic effects consistent with in vivo findings. In contrast, the clinically validated resistance delay and thus long-term synergy of targeted therapeutics in advanced melanoma models has been demonstrated using impedance technology. The results demonstrate limited clinical transferability of 2D/3D cancer cell line-based chemosensitivity data and underline the importance of in vivo-like direct patient-derived tissue for predictive drug studies. Our non-invasive and highly sensitive multidimensional impedance platform offers great potential for quantifying short- and long-term drug kinetics and synergies to identify the most effective drug combinations in advanced cancer models, thereby improving personalized drug development and treatment planning and ultimately, overall patient outcomes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Here we present a novel electrically switchable nanovalve array based on an intrinsic conductive polymer that has the capabilities to change its volume depending on its redox state. The polymer is created by anodic deposition of a sodium dodecylbenzenesulfonate (DBS)-doped polypyrrole (PPy). Optimization of the DBS-doped PPy layers revealed an actuatoric performance of up to 10% out of plane volume change. More interestingly, the electrochemical characterization revealed an actuatoric monostable polymer that could be used to fabricate nanovalve arrays that have a native opened state when no potential is applied and that can be closed when a reductive potential is applied. As a proof of concept, Atto488-labeled biotin (Biotin-Atto488) was used as a model compound and defined nanovalve arrays with nanopores in the range of 10 nm in diameter (opened state) were fabricated. Afterward, we were able to successfully prove the functionality of our nanovalve array by monitoring the flow-through rates of the Biotin-Atto488. More strikingly, we could demonstrate for the first time the robust and long-term stability of our nanovalve array without any performance loss for at least 72 h and retention capabilities of up to 90%. Furthermore, the demonstrated long-term stability was achieved under biocompatible conditions without the need of toxic dopant supplementation of the flow-through solution. Thus, our novel functional long-term stable nanovalve array offers the capabilities for practical applications.

Microreactors have gained increasing attention in their application toward continuous micro flow synthesis. An unsolved problem of continuous flow synthesis is the lack of techniques for continuous product purification. Herein, we present a micro free-flow electrophoresis device and accompanying setup that enables the continuous separation and purification of unlabeled organic synthesis products. The system is applied to the separation and purification of triarylmethanes. For imaging of the unlabeled analytes on-chip a novel setup for large area (3.6 cm 2 ) deep ultra violet excitation fluorescence detection was developed. Suitable separation conditions based on low conductivity electrophoresis buffers were devised to purify the product. With the optimized conditions, starting materials and product of the synthesis were well separated (R > 1.2). The separation was found to be very stable with relative standard deviations of the peak positions smaller than 3.5% over 15 min. The stable conditions enabled collection of the separated compounds, and purity of the product fraction was confirmed using capillary electrophoresis and mass spectrometry. This result demonstrates the great potential of free-flow electrophoresis as a technique for product purification or continuous clean-up in flow synthesis. Graphical Abstract Micro free-flow electrophoresis (μFFE) allows continuous separation and purification of small organic synthesis products. Enabled by a novel deep-UV imaging setup starting materials and product of a recently developed synthesis for triarylmethanes could be purified. Thereby demonstrating the potential of μFFE as continuous purification technique for micro flow synthesis.

For miniaturization and integration of chemical synthesis and analytics on small length scales, the development of complex lab-on-chip (LOC) systems is in the focus of many current research projects. While application specific synthesis and analytic modules and LOC devices are widely described, the combination and integration of different modules is intensively investigated. Problems for in-line processes such as solvent incompatibilities, e.g., for a multistep synthesis or the combination of an organic drug synthesis with a cell-based biological activity testing system, require a solvent exchange between serialized modules. Here, we present a continuously operating microfluidic solvent exchanger based on the principle of free-flow electrophoresis for miscible organic/aqueous fluids. We highlight a proof-of-principle and describe the working principle for the model compound fluorescein, where the organic solvent DMSO is exchanged against an aqueous buffer. The DMSO removal performance could be significantly increased to 95% by optimization of the microfluidic layout. Moreover, the optimization of the inlet flow ratio resulted in a minimized dilution factor of 5, and we were able to demonstrate that a reduction of the supporting instrumentation is possible without a significant decrease of the DMSO removal performance. Finally, the compatibility of the developed solvent exchanger for cell based downstream applications was proven. The impedimetric monitoring of HEK293A cells in a continuously operating microfluidic setup revealed no adverse effects of the residual DMSO after the solvent replacement. Our solvent exchanger device demonstrates the power of micro-free-flow electrophoresis not only as a powerful technique for separation and purification of compound mixtures but also for solvent replacement.

Lab-on-a-chip devices that combine, e.g. chemical synthesis with integrated on-chip analytics and multi-compartment organ-on-a-chip approaches, are a fast and attractive evolving research area. While integration of appropriate cell models in microfluidic setups for monitoring the biological activity of synthesis products or test compounds is already in focus, the integration of label-free bioelectronic analysis techniques is still poorly realized. In this context, we investigated the capabilities of impedance spectroscopy as a non-destructive real-time monitoring technique for adherent cell models in a microfluidic setup. While an initial adaptation of a microelectrode array (MEA) layout from a static setup revealed clear restrictions in the application of impedance spectroscopy in a microfluidic chip, we could demonstrate the advantage of a FEM simulation based rational MEA layout optimization for an optimum electrical field distribution within microfluidic structures. Furthermore, FEM simulation based analysis of shear stress and time-dependent test compound distribution led to identification of an optimal flow rate. Based on the simulation derived optimized microfluidic MEA, comparable impedance spectra characteristics were achieved for HEK293A cells cultured under microfluidic and static conditions. Furthermore, HEK293A cells expressing Y1 receptors were used to successfully demonstrate the capabilities of impedimetric monitoring of cellular alterations in the microfluidic setup. More strikingly, the maximum impedimetric signal for the receptor activation was significantly increased by a factor of 2.8. Detailed investigations of cell morphology and motility led to the conclusion that cultivation under microfluidic conditions could lead to an extended and stabilized cell-electrode interface.

Enzymes are the most effective catalysts for a broad range of difficult chemical reactions e.g. hydroxylation of non-activated C-H Bonds and stereoselective synthesis. Nevertheless, a lot of enzymes are not accessible for the biotechnological applications or industrial use. One reason is the prerequisite of expensive cofactors. In this context, we developed a bioelectrocatalytic analysis platform for the electrochemical and photonic quantification of the direct electron transfer from the electrode to redox enzymes and therefore, bypass the need of soluble cofactors that had to be continuously exchanged or regenerated. As reference enzyme, we chose cytochrome P450 BM3 that is restricted by NADPH dependence. We optimized the substrate spectrum for aromatic compounds by introduction of the triple mutation A74G/F87V/L188Q and established a sensitive fluorimetric product formation assay to monitor the enzymatic conversion of 7-ethoxycoumarine to 7-hydroxycoumarine. Gold and indium tin oxide electrodes were characterized with respect to surface morphology, charge-transfer resistance and P450 BM3 immobilization as well as activity. Using gold electrodes, no significant product formation by electrode mediated direct electron transfer could be detected. In contrast, P450 BM3 adsorbed on unmodified indium tin oxide electrodes revealed 36% activity by electrode mediated direct electron transfer in comparison to enzyme regeneration by NADPH. Since the reaction volumes are in the microliter range and upscaling of the measurement system is easily possible, our analysis platform is a useful tool for bioelectrocatalytic enzyme characterization and library screening based optimization for applications in the field of enzyme catalyzed chemical synthesis but also enzyme based fuel cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Due to the lack of appropriate cell models as well as automated electrophysiology monitoring technologies, the standardized identification of neurotoxic or protective effects in vitro remains a major problem in today's pharmaceutical ingredient development. Over the past few years, in vivo-like human pluripotent stem cell-derived neuronal networks have turned out to be a promising physiological cell source, if the establishment of robust and time-saving functional maturation strategies based on stable and expandable neural progenitor populations can be achieved. Here, we describe a multi-microelectrode array (MMEA)-based bioelectronics platform that was optimized for long-term electrophysiological activity monitoring of neuronal networks via field potential measurements. Differentiation of small molecule-based neuronal progenitors on MMEAs led to functional neurons within 15 days. More strikingly, these functional neuronal cultures could remain electrophysiologically stable on the MMEAs for more than four weeks. The observed electrophysiological properties correlated with the expression of typical neuron subtype markers and were further validated by specific neurotransmitter applications. With our established monitoring platform, we could show for the first time the long-term stability of the neural stem cell-like progenitor population to differentiate to electrophysiologically active dopaminergic neuronal networks for more than 80 passages. In conclusion, we provide a comprehensive long-term stable field potential monitoring platform based on stem cell-derived human neuronal networks that can be automated and up-scaled for standardized high-content screening applications e.g. in the field of neurotoxic and neuroprotective therapeutics identification.

Over the last decades, countless bioelectronic monitoring systems were developed for the analysis of cells as well as complex tissues. Most studies addressed the sensitivity and specificity of the bioelectronic detection method in comparison to classical molecular biological assays. In contrast, the up scaling as a prerequisite for the practical application of these novel bioelectronic monitoring systems is mostly only discussed theoretically. In this context, we developed a novel 384-multiwell microelectrode array (MMEA) based measurement system for the sensitive label-free real-time monitoring of neurodegenerative processes by impedance spectroscopy. With respect to the needs of productive screening systems for robust and reproducible measurements on high numbers of plates, we focused on reducing the critical contacting of more than 400 electrodes for a 384-MMEA. Therefore, we introduced an on top array of immersive counter electrodes that are individually addressed by a multiplexer and connected all measurement electrodes on the 384-MMEA to a single contact point. More strikingly, our novel approach provided a comparable signal stability and sensitivity similar to an array with integrated counter electrodes. Next, we optimized a SH-SY5Y cell based tauopathy model by introducing a novel 5-fold Tau mutation eliminating the need of artificial tauopathy induction. In combination with our novel 384-MMEA based measurement system, the concentration and time dependent neuroregenerative effect of the kinase inhibitor SRN-003-556 could be quantitatively monitored. Thus, our novel screening system could be a useful tool to identify and develop potential novel therapeutics in the field of Tau-related neurodegenerative diseases., (Copyright © 2016. Published by Elsevier B.V.)

In today's neurodevelopment and -disease research, human neural stem/progenitor cell-derived networks represent the sole accessible in vitro model possessing a primary phenotype. However, cultivation and moreover, differentiation as well as maturation of human neural stem/progenitor cells are very complex and time-consuming processes. Therefore, techniques for the sensitive non-invasive, real-time monitoring of neuronal differentiation and maturation are highly demanded. Using impedance spectroscopy, the differentiation of several human neural stem/progenitor cell lines was analyzed in detail. After development of an optimum microelectrode array for reliable and sensitive long-term monitoring, distinct cell-dependent impedimetric parameters that could specifically be associated with the progress and quality of neuronal differentiation were identified. Cellular impedance changes correlated well with the temporal regulation of biomolecular progenitor versus mature neural marker expression as well as cellular structure changes accompanying neuronal differentiation. More strikingly, the capability of the impedimetric differentiation monitoring system for the use as a screening tool was demonstrated by applying compounds that are known to promote neuronal differentiation such as the γ-secretase inhibitor DAPT. The non-invasive impedance spectroscopy-based measurement system can be used for sensitive and quantitative monitoring of neuronal differentiation processes. Therefore, this technique could be a very useful tool for quality control of neuronal differentiation and moreover, for neurogenic compound identification and industrial high-content screening demands in the field of safety assessment as well as drug development., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

The analysis of receptor activity, especially in its native cellular environment, has always been of great interest to evaluate its intrinsic but also downstream biological activity. An important group of cellular receptors are ion channels. Since they are involved in a broad range of crucial cell functions, they represent important therapeutic targets. Thus, novel analytical techniques for the quantitative monitoring and screening of biological receptor activity are of great interest. In this context, we developed an impedance spectroscopy-based label-free and non-invasive monitoring system that enabled us to analyze the activation of the transient receptor potential channel Vanilloid 1 (TRPV1) in detail. TRPV1 channel activation by capsaicin resulted in a reproducible impedance decrease. Moreover, concentration response curves with an EC 50 value of 0.9 μM could be determined. Control experiments with non TRPV1 channel expressing HEK cells as well as experiments with the TRPV1 channel blocker ruthenium red validated the specificity of the observed impedance decrease. More strikingly, through correlative studies with a cytoskeleton restructuring inhibitor mixture and equivalent circuit analysis of the acquired impedance spectra, we could quantitatively discriminate between the direct TRPV1 channel activation and downstream-induced biological effects. In summary, we developed a quantitative impedimetric monitoring system for the analysis of TRPV1 channel activity as well as downstream-induced biological activity in living cells. It has the capabilities to identify novel ion channel activators as well as inhibitors for the TRPV1 channel but could also easily be applied to other ion channel-based receptors.

Aggressive cancer entities like neuroblastoma and glioblastoma multiforme are still difficult to treat and have discouraging prognosis in malignant stage. Since each tumor has its own characteristics concerning the sensitivity towards different chemotherapeutics and moreover, can obtain resistance, the development of novel chemotherapeutics with a broad activity spectrum, high efficacy and minimum side effects is a continuous process. Sophisticated in vitro assays for comprehensive prediction of in vivo drug efficacy and side effects represent an actual bottleneck in the drug development process. In this context, we developed a novel in vitro 2D and 3D multiwell-multielectrode device for drug efficacy monitoring based on direct real-time impedance spectroscopy measurement in combination with our unique 96-well multielectrode arrays and microcavity arrays. For demonstration, we used three neuro- and glioblastoma cell lines that were cultured as monolayer and multicellular tumor spheroids for recapitulating in vivo conditions. Using our novel 96-well multielectrode array based system it was possible to detect time and concentration dependent responses concerning treatment with doxorubicin, etoposide and vincristine. While all tested chemotherapeutics revealed high potency for apoptosis induction in neuroblastoma cells, etoposide was ineffective for glioblastoma cell lines. Determination of IC50 values allowed us to compare drug efficacy in 2D and 3D culture models and moreover, revealed chemotherapeutic and tumor cell line specific activity patterns. These pharmacokinetic patterns are of great interest in the context of preclinical drug development. Thus, impedance spectroscopy based monitoring systems could be used for the fast in vitro based in vivo prediction of novel anti-tumor drugs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Stage III/IV melanoma remains incurable in most cases due to chemotherapeutic resistance. Thus, predicting and monitoring chemotherapeutic responses in this setting offer great interest. To overcome limitations of existing assays in evaluating the chemosensitivity of dissociated tumor cells, we developed a label-free monitoring system to directly analyze the chemosensitivity of undissociated tumor tissue. Using a preparation of tumor micro-fragments (TMF) established from melanoma biopsies, we characterized the tissue organization and biomarker expression by immunocytochemistry. Robust generation of TMF was established successfully and demonstrated on a broad range of primary melanoma tumors and tumor metastases. Organization and biomarker expression within the TMF were highly comparable with tumor tissue, in contrast to dissociated, cultivated tumor cells. Using isolated TMF, sensitivity to six clinically relevant chemotherapeutic drugs (dacarbazine, doxorubicin, paclitaxel, cisplatin, gemcitabine, and treosulfan) was determined by impedance spectroscopy in combination with a unique microcavity array technology we developed. In parallel, comparative analyses were performed on monolayer tumor cell cultures. Lastly, we determined the efficacy of chemotherapeutic agents on TMF by impedance spectroscopy to obtain individual chemosensitivity patterns. Our results demonstrated nonpredictable differences in the reaction of tumor cells to chemotherapy in TMF by comparison with dissociated, cultivated tumor cells. Our direct impedimetric analysis of melanoma biopsies offers a direct ex vivo system to more reliably predict patient-specific chemosensitivity patterns and to monitor antitumor efficacy., (©2014 American Association for Cancer Research.)

The enteric nervous system (ENS) poses the intrinsic innervation of the gastrointestinal tract and plays a critical role for all stages of postnatal life. There is increasing scientific and clinical interest in acquired or age-related gastrointestinal dysfunctions that can be manifested in diseases such as gut constipation or fecal incontinence. In this study, we sought to analyze age-dependent changes in the gene expression profile of the human ENS, particularly in the myenteric plexus. Therefore, we used the laser microdissection technique which has been proven as a feasible tool to analyze distinct cell populations within heterogeneously composed tissues. Full biopsy gut samples were prepared from children (4-12 months), middle aged (48-58 years) and aged donors (70-95 years). Cryosections were histologically stained with H&E, the ganglia of the myenteric plexus identified and RNA isolated using laser microdissection technique. Quantitative PCR was performed for selected neural genes, neurotransmitters and receptors. Data were confirmed on protein level using NADPH-diaphorase staining and immunohistochemistry. As result, we demonstrate age-associated alterations in site-specific gene expression pattern of the ENS. Thus, in the adult and aged distal parts of the colon a marked decrease in relative gene expression of neural key genes like NGFR, RET, NOS1 and a concurrent increase of CHAT were observed. Further, we detected notable regional differences of RET, CHAT, TH, and S100B comparing gene expression in aged proximal and distal colon. Interestingly, markers indicating cellular senescence or oxidative stress (SNCA, CASP3, CAT, SOD2, and TERT) were largely unchanged within the ENS. For the first time, our study also describes the age-dependent expression pattern of all major sodium channels within the ENS. Our results are in line with previous studies showing spatio-temporal differences within the mammalian ENS.

For selective killing of tumor cells, there are many novel and promising therapeutic approaches like immunotoxins. However, on the long way to clinical application, especially in vitro approved biologicals often fail due to loss of target sensitivity and efficacy in vivo. This is mostly explained with degradation or penetration disability in vivo. Although, these problems are well known, until today, there are no in vitro systems for reliable monitoring and quantification of therapeutic efficacy in 3D tumor models and the direct comparison to results from 2D models. In this context, we developed a combined label-free impedimetric monitoring system using our self-developed planar interdigital electrode arrays and our unique microcavity array technology. Therefore, we could demonstrate the time and concentration dependent monitoring and quantification of therapeutic efficacy in a 2D and 3D mamma carcinoma model. In detail, we synthesized a novel modular immunotoxin B3(dsFv)-PE38 (B3-PE38) in which the antibody fragment and the protein toxin are polyionic linked together. We compared the efficacy of the immunotoxin B3-PE38, the toxin E8C-PE38 (PE38) and the small molecule chemotherapeutic paclitaxel. The impedimetric screening revealed the highest cytotoxicity for the immunotoxin B3-PE38 in the 2D model. More strikingly, the immunotoxin efficacy was substantially higher in the 3D model when compared to PE38 and paclitaxel even though having a considerably lower penetration capability than paclitaxel. So our novel impedimetric monitoring system offers the comparative efficacy quantification of novel therapeutics in 2D and 3D in vitro tumor models., (© 2013 Published by Elsevier B.V.)

Until today, brain tumors especially glioblastoma are difficult to treat and therefore, results in a poor survival rate of 0-14% over five years. To overcome this problem, the development of novel therapeutics as well as optimization of neurosurgical procedures to remove the tumor tissue are subject of intensive research. The main problem of the tumor excision, as the primary clinical intervention is the diffuse infiltration of the tumor cells in unaltered brain tissue that complicates the complete removal of residual tumor cells. In this context, we are developing novel approaches for the label-free discrimination between tumor tissue and unaltered brain tissue in real-time during the surgical process. Using our impedance spectroscopy-based measurement system in combination with flexible microelectrode arrays we could successfully demonstrate the discrimination between a C6-glioma and unaltered brain tissue in an in vivo rat model. The analysis of the impedance spectra revealed specific impedance spectrum shape characteristics of physiologic neuronal tissue in the frequency range of 10-500 kHz that were significantly different from the tumor tissue. Moreover, we used an adapted equivalent circuit model to get a deeper understanding for the nature of the observed effects. The impedimetric label-free and real-time discrimination of tumor from unaltered brain tissue offers the possibility for the implementation in surgical instruments to support surgeons to decide, which tissue areas should be removed and which should be remained., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Unexpected adverse effects on the cardiovascular system remain a major challenge in the development of novel active pharmaceutical ingredients (API). To overcome the current limitations of animal-based in vitro and in vivo test systems, stem cell derived human cardiomyocyte clusters (hCMC) offer the opportunity for highly predictable pre-clinical testing. The three-dimensional structure of hCMC appears more representative of tissue milieu than traditional monolayer cell culture. However, there is a lack of long-term, real time monitoring systems for tissue-like cardiac material. To address this issue, we have developed a microcavity array (MCA)-based label-free monitoring system that eliminates the need for critical hCMC adhesion and outgrowth steps. In contrast, feasible field potential derived action potential recording is possible immediately after positioning within the microcavity. Moreover, this approach allows extended observation of adverse effects on hCMC. For the first time, we describe herein the monitoring of hCMC over 35 days while preserving the hCMC structure and electrophysiological characteristics. Furthermore, we demonstrated the sensitive detection and quantification of adverse API effects using E4031, doxorubicin, and noradrenaline directly on unaltered 3D cultures. The MCA system provides multi-parameter analysis capabilities incorporating field potential recording, impedance spectroscopy, and optical read-outs on individual clusters giving a comprehensive insight into induced cellular alterations within a complex cardiac culture over days or even weeks.

Alzheimer's disease (AD) and other tauopathies comprise death of cell bodies, synapses and neurites but there is surprising little knowledge of the temporal sequence and the causal relationships among these events. Here, we present a novel biosensoric approach to monitor retrograde neurite degeneration before cell death occurs. We induced tau hyperphosphorylation in organotypic hippocampal slice cultures (OHSC) and applied marker-independent real-time electrical impedance spectroscopy (EIS) for cellular real-time pathology monitoring. Using this approach, we were able to define two distinct phases of neurite degeneration, first a rapid swelling of axonal processes that manifests itself in relative impedance above control levels followed by a slower phase of collapse and subsequent fragmentation indicated by decreased relative impedance below control levels. Initial axon swelling is strictly dose-dependent and swelling intensity correlates with second phase impedance decrease implicating a causative link between both degenerative mechanisms. Moreover, suppressing tau hyperphosphorylation by kinase inhibition nearly prevented both phases of axon degeneration. Our findings demonstrate that the temporal sequence of tau-triggered neurite degeneration can be directly visualized by EIS-based, non-invasive and label-free monitoring. We therefore suggest this approach as a powerful extension of high content applications to study mechanisms of neurite degeneration and to exploit therapeutic options against AD and tau-related disorders., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy.With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research.

Electrochemical biosensors allow simple, fast and sensitive analyte detection for various analytical problems. Especially immunosensors are favourable due to specificity and affinity of antigen recognition by the associated antibody. We present a novel electrode array qualified for parallel analysis and increased sample throughput. The chip has nine independent sample chambers. Each chamber contains a circular gold working electrode with a diameter of 1.9 mm that is surrounded by a ring-shaped auxiliary electrode with a platinum surface. The corresponding silver/silver chloride reference electrodes are embedded in a sealing lid. The chip is open to the full range of electrochemical real-time detection methods. Among these techniques, impedance spectroscopy is an attractive tool to detect fast and label-free interfacial changes originating from the biorecognition event at the electrode surface. The capabilities of the novel electrode array are demonstrated using the example of tumour marker tenascin C detection. This glycoprotein of the extracellular matrix is expressed in cancerous tissues, especially in solid tumours such as glioma or breast carcinoma. Electrodes covered with specific antibodies were exposed to tenascin C containing samples. Non-occupied binding sites were identified using a secondary peroxidase-conjugated antibody that generated an insoluble precipitate on the electrode in a subsequent amplification procedure. The charge transfer resistance obtained from impedimetric analysis of ferri-/ferrocyanide conversion at the electrode served as analytic parameter. This assay detected 14 ng (48 fmol) tenascin C that is sufficient for clinical diagnostics. The electrode surface could be regenerated at least 20-fold without loss of its analytical performance.

Transient receptor potential (TRP) channels are non-selective ion channels permeable to cations including Na(+), Ca(2+) and Mg(2+). They play a unique role as cellular sensors and are involved in many Ca(2+)-mediated cell functions. Failure in channel gating can contribute to complex pathophysiological mechanisms. Dysfunctions of TRP channels cause diseases but are also involved in the progress of diseases. We present a novel method to analyse chemical compounds as potential activators or inhibitors of TRP channels to provide pharmaceutical tools to regulate channel activity for disease control. Compared to common methods such as patch clamp or Ca(2+) imaging, the presented impedance assay is automatable, experimental less demanding and not restricted to Ca(2+) flow. We have chosen TRPA1 from the TRPA ('ankyrin') family as a model channel which was stimulated by allyl isothiocyanate (AITC). HEK293 cells stably transfected with human TRPA1 cDNA were grown on microelectrode arrays. Confluent cell layers of high density were analysed. Impedance spectra of cell-covered and non-covered electrodes yielded a cell-specific signal at frequencies between 70 and 120 kHz. Therefore, 100 kHz was chosen to monitor TRPA1 activity thereupon. An average impedance decrease to about 70% of its original value was observed after application of 10 μM AITC indicating an increased conductance of the cell layer mediated by TRPA1. Transfected cells pretreated with 10 μM of inhibitor ruthenium red to prevent channel conductance, as well as control cells lacking TRPA1, showed no impedance changes upon AITC stimuli demonstrating the specificity of the novel impedance assay., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Cardiovascular diseases represent the most common cause of death in industrialized countries. In this context vascular smooth muscle cells (SMCs) are a major key player that is involved in pathological processes like hypertension and atherosclerosis. Therefore the pharmaceutical industry is intensively investigated in developing non-destructive and label-free monitoring techniques for a quantitative detection of SMC characteristics in the field of active pharmaceutical development as well as clinical diagnostics. Hence, we developed a novel multiwell interdigital electrode sensor-array in standardized ANSI 96-well layout. Through optimization of electrode geometry and material as well as passivation/adhesion-layer we obtained a novel biohybrid chip for the sensitive and quantitative detection of SMC contractility as well as relaxation via impedance spectroscopy. For the validation of our multiwell sensor-array we established a SMC culture model derived from primary cells that is switchable from a non-contractile pathological to a functional contractile phenotype. Using the reference compounds acetylcholine (ACh) and amlodipine, we could quantify SMC contraction by an impedance decrease to 40% while SMC relaxation was detectable by an impedance increase to 110%. More strikingly we could monitor aging of the isolated SMC which arose by an attenuated contractility over successive passaging. Demonstrating the performance of our self-developed multiwell sensor-array based impedance measurement setup we provide a suitable sensor-array coupled cell model to study the mechanisms that activated SMCs undergo in response to inflammatory mediators or vessel injury.

Herewith we developed a novel 3D in vitro Alzheimer's disease (AD) model, based on the human neuroblastoma cell line SH-SY5Y, which is well differentiated without the application of any agents. Furthermore AD-like pathological neurodegeneration can be induced by okadaic acid (OA) mediated hyperphosphorylation of the microtubule associated protein tau. Moreover, we established stable "rapid tauopathy cell lines" expressing additional EGFP-fused (enhanced green fluorescent protein) wildtype or a pathology-promoting mutant tau variant (P301L) by lentiviral transduction. For the sensitive and feasible quantitative detection of pathological effects on neuronal 3D-cultures by electrochemical impedance spectroscopy (EIS) we optimized and redesigned a microcavity array (MCA). The cellular contribution to impedance could be increased by the factor of 2.5 and the variance decreased by 40%. Using our optimized MCA and impedance measurement setup we were able to detect quantitatively an OA concentration- and time-dependent decrease of the impedance in 3D SH-SY5Y cultures. Moreover, we were able to detect and quantify distinct, AD-related effects triggered by tau-mutant (P301L) expression and hyperphosphorylation in our organotypic 3D-cultures with the help of impedance spectroscopy., (Copyright 2010 Elsevier B.V. All rights reserved.)

An overall objective of pharmaceutical research is the controlled release or delivery of drugs at the biological target site in a therapeutically and pharmacodynamically optimal amount. In relation to "intelligent" drug delivery, several basic aspects are important, i.e., release of active pharmaceutical ingredients from the formulation, transport to and penetration across biological barriers, and subsequent biotransformation depending on a controlled release process. Future development of advanced and/or controlled drug releasing systems, e.g. polymeric or particulate drug targeting systems, nano-carbon tube related and/or nano-pillar based drug release, or electronically mediated molecule delivery, is expected to take advantage of progress in molecular cell biology, cell and tissue engineering, membrane nano-biophysics, and bioelectronic properties (Bramstedt et al. 2005; Gardner et al. 2006). In this chapter novel aspects of the development of innovative drug delivery systems described and are categorized into polymeric, lipid-based or electronically mediated delivery systems (De la Heras et al. 2004).

Tauopathies such as Alzheimer's disease (AD) belong to the group of neurodegenerative diseases that are characterised by hyperphosphorylation of the protein tau. Hyperphosphorylation of tau is one of the salient events leading to neuronal cytotoxicity and cognitive impairments. In this context, inhibition of tau hyperphosphorylation by specific tau kinase inhibitors can provide an excellent drug target for the treatment of AD and other tau-related neurodegenerative diseases. To improve the identification, optimisation and validation during the high-cost hit-to-lead cycle of AD drugs, we established a fast and sensitive label-free technique for testing the efficacy of tau kinase inhibitors in vitro. Here, we report for the first time that microelectrode-based impedance spectroscopy can be used to detect the pathological risk potential of hyperphosphorylated tau in the human neuroblastoma cell line SH-SY5Y. Our findings provide a novel real-time recording technique for testing the efficiency of tau kinase inhibitors or other lead structures directed to tau hyperphosphorylation on differentiated SH-SY5Y cells.

Heart diseases represent the most common cause of death in industrialised countries. For this reason target identification and development of novel anti-target drugs are in the focus of pharmaceutical industry. Especially cardiac infarct is a topical field of research. A bottleneck in today's long-duration and high-cost drug development is the lack of fast, label-free and cell-based high throughput/high content screening (HTS/HCS) assays for bridging the gap between cell-free screening and animal experiments. Here, we report for the first time on an in vitro cardiac ischemic model, where pathological consequences of simulated cardiac infarct can be detected quantitatively by microelectrode array-based impedance spectroscopy. Using the contractile HL-1 cell line and defined ischemic conditions we were able to develop a standardised and reproducible pathologic model. We characterised and verified the HL-1 based ischemic model by apoptosis and proliferation assays as well as immunochemical analysis of cell-cell junctions. We showed that the observed cell and biomolecular effects correspond with results obtained by impedance spectroscopy. Functionality of the impedimetric assay was demonstrated by real-time detection of reduced pathological effects due to application of the selective Rac1 inhibitor NCS23766. Numerical analysis by means of an equivalent circuit allowed the quantification of changes in resistance and capacitance of the adherent cell layer after ischemic treatment and application of NSC23766 as drug model. Our findings provide a novel cell-based real-time screening system for testing drug candidates against cardiac infarct and its implications.

Unlabelled: We describe the use of rotary cultures (72 rpm) as an excellent method for generating spheroids from dispersed bovine granulosa cells (GC). The GC spheroids were symmetrical (diameter between 100 and 200 microm), easily accessible, and could be obtained at high yields. On day one, the spheroids showed a two-layered outer zone of cells that stained lighter than the inner zone in semi-thin sections. Bromodeoxyuridine (BrdU) uptake was frequent and randomly distributed. By day two, a striking decrease in BrdU uptake was noted. Apoptotic bodies appeared up to day four, as did TUNEL and propidium iodide labelled dead cells. At that time, the inner zone contained cells with large-sized vacuoles and the core was amorphous. The large-sized vacuoles were identified at the ultrastructural level and represented autophagosomes and autophagolysosomes that were in different stages of development. Surprisingly, conspicuous signs of cell death were accompanied by an increase in spontaneous luteinization compared to conventional stationary cultures. We detected high levels of progesterone (immunoassay) accompanied by high levels of the proteins and enzymes relevant for steroidogenesis (StAR, P450scc, 3beta-HSD by immunoblot and immunohistochemistry, respectively)., Conclusions: Concomitant to cell death, GC spheroids augment progesterone synthesis. The GC spheroids provide an ideal model for studying steroidogenesis coupled to programmed cell death at the level of the mitochondria.

Sensorchip based impedance spectroscopy can detect inhibitory effects of human neuropeptide Y (hNPY) on living cells in a non-invasive labelling free way in real time without the need of supporting reagents. Since the discovery that neoplasmatic transformations in breast cancer are correlated with a change of the receptor subtype expression of hNPY in the affected tissue, the hNPY receptor-ligand system has come to the fore of cancer research. Today there are different methods detecting hNPY receptor interactions like fluorescent and radioactive labelling or detecting hNPY-pathway activation like cyclic adenosine monophosphate (cAMP) and G protein-coupled receptor (GPCR)-assays. For all these assays it is necessary to either label related proteins with additional substances, which can affect the nature state of the cell, or the need of producing cell lysate which allows only a snapshot of the investigated cells. To overcome these problems we established a new method to detect hNPY-receptor interactions. Therefore, we monitor the complex electric resistance (impedance) of cells attached to a microelectrode over a wide frequency range. Cell alterations are detected as changes in the impedance spectra. After application of the adenylyl cyclase-stimulating reagent forskolin, impedance is decreased at 5 kHz frequency within minutes. This effect can be inhibited by preincubating the cells with hNPY for a time range of 20 min. The inhibitory effect of hNPY can be washed out and the same cells can be stimulated by forskolin again.

The glial cell line-derived neurotrophic factor (GDNF) family consists of the four ligands GDNF, neurturin (NRTN), artemin and persephin, which bind to the four co-receptors GDNF family receptor alpha1-4 and control through the activation of the receptor tyrosin kinase Ret several developmental processes. The purpose of this study was to analyse the expression and the influence of NRTN in the developing retina. We used retinospheres, a three-dimensional model system of the developing chicken retina. The expression of NRTN and the GDNF family receptor alpha 2 increased during development. Furthermore, expression was comparable in retinae and retinospheres. Analysis of signalling pathways influenced by NRTN in retinospheres showed activation of phosphatidylinositol-3 kinase and mitogen-activated protein kinase (MAPK). Activation of MAPK could be localised in cells of the innermost rows of the inner nuclear layer which were predominantly acetylcholinesterase-positive cells. Exogenous application of NRTN increased the amount of acetylcholinesterase-positive cells within the retinospheres at late culture stages. Additionally, we could show that Müller glia cells did not express the GFRalpha2 receptor and were probably not involved in NRTN signalling. Therefore, we conclude that NRTN directly participates in regulatory processes concerning the differentiation of acetylcholinesterase-positive cells in the chicken retina.

Persephin (PSPN), a member of the glial cell line-derived neurotrophic factor family, and its implication in the retina is not well understood but might be an interesting therapeutic target for degenerative diseases. Although, PSPN is lost in the chicken during evolution, its target, the GDNF family receptor alpha 4 (GFRalpha4), is still expressed in a temporal and spatial pattern in the developing retina. We used this "knockout-precondition" to study the bioactivity and the effect of exogenous PSPN application and subsequent GFRalpha activation during retinal development in vitro without impairments of endogenous PSPN. Retinospheres, derived from dissociated chicken retina of embryonic day 6, were treated with PSPN and intracellular signalling was monitored. Additionally, PSPN was added during cultivation of the retinospheres and immunhistochemical stainings and Western blotting were performed to evaluate changes in proliferation, apoptosis and differentiation. Exogenous applied PSPN enhanced phosphatidylinositol-3-kinase (PI-3K) signalling and decreased signalling of mitogen-activated protein kinases (MAPK). Most importantly early retinal proliferation was enhanced and glutamine synthetase expression was decreased whereas differentiation of major retinal cell types was not changed. In contrast to GDNF, PSPN is exclusively influencing early progenitors whereas differentiation is not effected and seems to be regulated through PSPN-independent mechanisms. Since the binding site of PSPN and therefore the target of potential therapeuticals, is well conserved among species and is with high probability not able to bind other members of the GDNF-family, these results might be assigned to other species including mammals and humans.

Close to realistic responses to anti-cancer drugs are not adequately provided in monolayer or single cells assays. 3-dimensional multicellular cultures (spheroids) mimicking in vivo-like conditions are established as cell biological models for microtumors/metastases. For a non-invasive real-time monitoring of the electrical parameters of such spheroid cultures we designed, fabricated and tested a 3D multifunctional electrode-based microcavity array. In a non-adherent assay acute tests with tumor spheroids were done maintaining their spherical shape and cellular arrangement. The sensor chip with 15 individual square microcavities containing four gold electrodes each was used for impedance spectroscopy to analyze the tissue models in terms of morphological and structural changes. Cell type specific differences in the spectra and varying responses to several anti-tumor drugs were found. Further development of the prototype will provide a promising tool for the use in pharmacological high-throughput studies.

Multicellular tumour spheroids that mimic a native cellular environment are widely used as model systems for drug testing. To study drug effects on three-dimensional cultures in real-time we designed and fabricated a novel type of sensor chip for fast, non-destructive impedance spectroscopy and extracellular recording. Precultured spheroids are trapped between four gold electrodes. Fifteen individual 100microm deep square microcavities with sizes from 200 to 400microm allow an optimised positioning during the measurement. Although apoptosis was induced in human melanoma spheroids by Camptothecin (CTT), treated cultures did not show disintegration but displayed increased impedance magnitudes compared to controls after 8h resulting from an altered morphology of the outer cells. Contractions in cardiomyocyte spheroids were monitored when the innovative chip was used for recording of extracellular potentials. The silicon-based electrode array is used as an acute test system for the monitoring of any kind of 3D cell cultures. Since no adherence of cells or labelling is necessary the multifunctional sensor chip provides a basis for improved drug development by high content screenings with reduced costs and assay times. Additional improvements for parallel testing of different substances on one chip are presented.

Glycogen is the major energy reserve in neural tissues including the retina. A key-enzyme in glycogen metabolism is glycogen phosphorylase (GP) which exists in three differentially regulated isoforms. By applying isozyme-specific antibodies it could be demonstrated that the GP BB (brain), but not the GP MM (muscle) isoform is expressed in the chicken retina in neuronal and glial (Müller) cells. In the embryonic chicken retina, GP showed a development-dependent expression pattern. Double-labeling experiments with cell type-specific antibodies revealed that GP is expressed in various layers of the retina some of which, e.g., the photoreceptor inner segments, are known to be sites of high energy consumption. This suggests important roles of GP BB, and therefore glycogen, in early differentiation, spontaneous wave generation and in formation and stabilization of synapses.

For a feasible and cost-effective impedance measurement of cellular alterations in real-time, we combined commercially available microelectrode arrays (MEAs), consisting of 60 microelectrodes, with a conventional impedance analyzer. For proof of principle, a breast carcinoma cell line (MCF-7) was cultured on MEAs, and cellular alterations were measured by impedance spectroscopy at a frequency ranging from 10 Hz to 1 MHz. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA) at different concentrations to activate protein kinase C (PKC)-mediated extra- and intracellular changes. By addition of 0.03 microM PMA, an increase of the relative impedance (Z(rel)) was observed after 10 min with a maximum at 1 kHz. Moreover a gradual elevation of the impedance was measured 60 min after stimulation with PMA. If 0.3 microM PMA was applied, the maximal amplitude of the relative impedance after 60 min shifted from 1 kHz (0.03 microM PMA) to 150 Hz. Subsequently, the impedance was further increased up to 90 min after PMA application, after which the impedance reduced after 240 min. Since we could use MEAs for at least 10 times without affecting the sensitivity, our study revealed that commercially available MEAs comprising nanocolumnar titanium nitrite electrodes are suitable microstructures for a highly reproducible and cost-effective multisite measurement of intracellular processes by impedance spectroscopy.

Purpose: To determine the role of glial cell line-derived neurotropic factor family receptor alpha 4 (GFRalpha4) during retinogenesis in a three-dimensional histiotypic in vitro model of the embryonic chicken retina., Methods: Retinal spheres were cultured from dissociated 6-day-old chicken retina under permanent rotation and transfected with GFRalpha4 siRNA at culture day 2. Alterations on proliferation, apoptosis, and differentiation were determined by semiquantitative RT-PCR, in situ hybridization, and immunohistochemistry after 24, 48, and 72 hours., Results: In contrast to control cultures, retinal spheres transfected with GFRalpha4 siRNA showed reduced GFRalpha4 mRNA expression of only 38% after 24 hours, 3% after 48 hours, and 5% after 72 hours. Based on the suppression of GFRalpha4, a decline in proliferating cells from 10% to 4.8% even after 24 hours and a reduction of sphere size by up to 25% at later culture stages were observed. Moreover, the number of Pax 6-positive amacrine, ganglion, and horizontal cells was significantly decreased from 36% to 16% in GFRalpha4 siRNA-transfected retinal spheres 72 hours after transfection. Additionally, GFRalpha4 gene silencing affected the development of different types of photoreceptors, as revealed by a significant decrease of blue opsin mRNA expression from 29% to 2%, whereas green opsin mRNA and the number rho4D2-positive photoreceptors were significantly increased., Conclusions: These data showed for the first time that GFRalpha4 plays an essential role in regulating, at least in vitro, the development and differentiation of various cell types during retinogenesis.

The goal of this study was to establish a reliable three-dimensional culture system for the mammalian retina that allows the analysis of retinal function and dysfunction. To produce three-dimensional retinal tissues in vitro, dissociated retinal cells of neonatal rats were maintained in culture dishes on a self-made orbital shaker. On the basis of well-defined rotation conditions, dissociated free-floating cells reaggregate in the center of the culture dish to form a multicellular cluster. Subsequently, cells begin to proliferate, whereby they form spherelike retinal tissues that grow to a size of 180-210 microm. Immunohistochemical characterization of mature retinal spheres revealed the presence of ganglion cells, amacrine cells, Müller cells, and rod photoreceptors, which are arranged in different retina-like layers. Although a small number of cells undergo programmed cell death, retinal spheres remain viable for at least 35 days in culture as revealed by fluorescein diacetate and TUNEL staining. Because most biological processes involved in tissue organization such as proliferation, differentiation, apoptosis, and survival are also observable in retinal spheres, the presented novel mammalian three-dimensional culture system is not only an outstanding model for basic research but may also be of great benefit for stem cell tissue engineering and the pharmaceutical industry.

The retinal ciliary margin is particularly relevant for the correct generation and regeneration of vertebrate retinae, since pluripotent stem cells are located there throughout development, and--at least in some species--even until adult stages. Our aim was to identify factors (genes) which are involved in processes of proliferation and differentiation in the developing chicken retina. Reverse transcription-polymerase chain reaction differential display was used to identify genes that were differentially expressed in chick central and peripheral embryonic retina. Candidate genes analyzed through sequencing and database searches were confirmed by Northern blot analysis and histochemistry. A series of differentially expressed genes were detected, including a neuronal cell adhesion molecule, an esterase, and homeobox gene products. One of the sequenced products was identified as subunit I of cytochrome-c oxidase (COX-1), an enzyme which is central to energy metabolism and particularly relevant for developing nervous systems. Northern blot analysis confirmed its up-regulation in the chick peripheral retina, being maximal at embryonic day 7. In the retinal pigmented epithelium its expression is lower than in the retinal periphery but higher than in central retina. COX histochemistry revealed distinct laminar patterns in central retina, but also an elevated level of activity in the peripheral retina throughout development. These data not only show that the developing ciliary margin of the chick retina has high energy requirements, but also indicate that COX-1 could play essential roles in developing cells and in stem cells of the eye periphery.