Novel Multiparametric Bioelectronic Measurement System for Monitoring Virus-Induced Alterations in Functional Neuronal Networks.

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.

Amphetamine increases vascular permeability by modulating endothelial actin cytoskeleton and NO synthase via PAR-1 and VEGF-R.

Abuse of amphetamine-type stimulants is linked to cardiovascular adverse effects like arrhythmias, accelerated atherosclerosis, acute coronary syndromes and sudden cardiac death. Excessive catecholamine release following amphetamine use causes vasoconstriction and vasospasms, over time leading to hypertension, endothelial dysfunction or even cardiotoxicity. However, immediate vascular pathomechanisms related to amphetamine exposure, especially endothelial function, remain incompletely understood and were analyzed in this study. Pharmaco-pathological effects of acute d-amphetamine-sulfate (DAM) were investigated ex vivo using contraction-force measurements of rat carotid artery rings and in vitro using label-free, real-time electrochemical impedance spectroscopy (EIS) on endothelial and smooth muscle cells. Specific receptor and target blocking was used to identify molecular targets and to characterize intracellular signaling. DAM induced vasodilation represented by 29.3±2.5% decrease in vascular tone (p<0.001) involving vascular endothelial growth factor receptor (VEGF-R) and protease activated receptor 1 (PAR-1). EIS revealed that DAM induces endothelial barrier disruption (-75.9±1.1% of initial cellular impedance, p<0.001) also involving VEGF-R and PAR-1. Further, in response to DAM, Rho-associated protein kinase (ROCK) mediated reversible contraction of actin cytoskeleton resulting in endothelial barrier disruption. Dephosphorylation of Serine1177 (-50.8±3.7%, p<0.001) and Threonine495 (-44.8±6.5%, p=0.0103) of the endothelial NO synthase (eNOS) were also observed. Blocking of VEGF-R and PAR-1 restored baseline eNOS Threonine495 phosphorylation. DAM induced vasodilation, enhanced vascular permeability and actin cytoskeleton contraction and induced eNOS hypophosphorylation involving VEGF-R, PAR-1 and ROCK. These results may contribute to a better understanding of severe adverse cardiovascular effects in amphetamine abuse.

Novel high-dense microelectrode array based multimodal bioelectronic monitoring system for cardiac arrhythmia re-entry analysis.

In recent decades, significant progress has been made in the treatment of heart diseases, particularly in the field of personalized medicine. Despite the development of genetic tests, phenotyping and risk stratification are performed based on clinical findings and invasive in vivo techniques, such as stimulation conduction mapping techniques and programmed ventricular pacing. Consequently, label-free non-invasive in vitro functional analysis systems are urgently needed for more accurate and effective in vitro risk stratification, model-based therapy planning, and clinical safety profile evaluation of drugs. To overcome these limitations, a novel multilayer high-density microelectrode array (HD-MEA), with an optimized configuration of 512 sensing and 4 pacing electrodes on a sensor area of 100 mm2, was developed for the bioelectronic detection of re-entry arrhythmia patterns. Together with a co-developed front-end, we monitored label-free and in parallel cardiac electrophysiology based on field potential monitoring and mechanical contraction using impedance spectroscopy at the same microelectrode. In proof of principle experiments, human induced pluripotent stem cell (hiPS)-derived cardiomyocytes were cultured on HD-MEAs and used to demonstrate the sensitive quantification of contraction strength modulation by cardioactive drugs such as blebbistatin (IC50 = 4.2 µM), omecamtiv and levosimendan. Strikingly, arrhythmia-typical rotor patterns (re-entry) can be induced by optimized electrical stimulation sequences and detected with high spatial resolution. Therefore, we provide a novel cardiac re-entry analysis system as a promising reference point for diagnostic approaches based on in vitro assays using patient-specific hiPS-derived cardiomyocytes.

Microcavity well-plate for automated parallel bioelectronic analysis of 3D cell cultures.

Three-dimensional (3D) in vitro cell culture models serve as valuable tools for accurately replicating cellular microenvironments found in vivo. While cell culture technologies are rapidly advancing, the availability of non-invasive, real-time, and label-free analysis methods for 3D cultures remains limited. To meet the demand for higher-throughput drug screening, there is a demanding need for analytical methods that can operate in parallel. Microelectrode systems in combination with microcavity arrays (MCAs), offer the capability of spatially resolved electrochemical impedance analysis and field potential monitoring of 3D cultures. However, the fabrication and handling of small-scale MCAs have been labour-intensive, limiting their broader application. To overcome this challenge, we have established a process for creating MCAs in a standard 96-well plate format using high-precision selective laser etching. In addition, to automate and ensure the accurate placement of 3D cultures on the MCA, we have designed and characterized a plug-in tool using SLA-3D-printing. To characterize our new 96-well plate MCA-based platform, we conducted parallel analyses of human melanoma 3D cultures and monitored the effect of cisplatin in real-time by impedance spectroscopy. In the following we demonstrate the capabilities of the MCA approach by analysing contraction rates of human pluripotent stem cell-derived cardiomyocyte aggregates in response to cardioactive compounds. In summary, our MCA system significantly expands the possibilities for label-free analysis of 3D cell and tissue cultures, offering an order of magnitude higher parallelization capacity than previous devices. This advancement greatly enhances its applicability in real-world settings, such as drug development or clinical diagnostics.

Integration of Impedimetric Sensors for In Situ Electrochemical Impedance Spectroscopy in Free-Flow Electrophoresis Applications in Lab-on-Chip Systems.

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.

Neuronal and glial cell co-culture organization and impedance spectroscopy on nanocolumnar TiN films for lab-on-a-chip devices.

Lab-on-a-chip devices, such as multielectrode arrays (MEAs), offer great advantages to study function and behavior of biological cells, such as neurons, outside the complex tissue structure. Nevertheless, in vitro systems can only succeed if they represent realistic conditions such as cell organization as similarly found in tissues. In our study, we employ a co-culture system of neuron-like (SH-SY5Y) and glial-like (U-87 MG) cells with various neuron-glial ratios to model different brain regions with different cellular compositions in vitro. We find that cell behavior in terms of cellular organization, as well as proliferation, depends on neuron-glial cell ratio, as well as the underlying substrate material. In fact, nanocolumnar titanium nitride (TiN nano), which exhibits improved electric properties for neural recording on MEA, shows improved biocompatible features compared to indium tin oxide (ITO). Moreover, electrochemical impedance spectroscopy experiments allow us to monitor cellular processes label-free in real-time over several days with multielectrode arrays. Additionally, electrochemical impedance experiments reveal superiority of TiN with nanocolumnar surface modification in comparison with ITO. TiN nano exhibits enhanced relative cell signals and improved signal-to-noise ratio, especially for smaller electrode sizes, which makes nanocolumnar TiN a promising candidate for research on neural recording and stimulation.

Kinase Inhibition by PKC412 Prevents Epithelial Sheet Damage in Autosomal Dominant Epidermolysis Bullosa Simplex through Keratin and Cell Contact Stabilization.

Epidermolysis bullosa simplex (EBS) is a severe and potentially life-threatening disorder for which no adequate therapy exists. Most cases are caused by dominant sequence variations in keratin genes K5 or K14, leading to the formation of cytoplasmic keratin aggregates, profound keratinocyte fragility, and cytolysis. We hypothesized that pharmacological reduction of keratin aggregates, which compromise keratinocyte integrity, represents a viable strategy for the treatment of EBS. In this study, we show that the multikinase inhibitor PKC412, which is currently in clinical use for acute myeloid leukemia and advanced systemic mastocytosis, reduced keratin aggregation by 40% in patient-derived K14.R125C EBS-associated keratinocytes. Using a combination of epithelial shear stress assay and real-time impedance spectroscopy, we show that PKC412 restored intercellular adhesion. Molecularly, global phosphoproteomic analysis together with immunoblots using phosphoepitope-specific antibodies revealed that PKC412 treatment altered phosphorylated sites on keratins and desmoplakin. Thus, our data provide a proof of concept to repurpose existing drugs for the targeted treatment of EBS and showcase how one broad-range kinase inhibitor reduced keratin filament aggregation in patient-derived EBS keratinocytes and the fragility of EBS cell monolayers. Our study paves the way for a clinical trial using PKC412 for systemic or local application in patients with EBS.

Multielectrode biosensor chip for spatial resolution screening of 3D cell models based on microcavity arrays.

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.

Novel PMMA based 96-well microelectrode arrays for bioelectronic high throughput monitoring of cells in a live mode.

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.

Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester Hydrolase.

Earth is flooded with plastics and the need for sustainable recycling strategies for polymers has become increasingly urgent. Enzyme-based hydrolysis of post-consumer plastic is an emerging strategy for closed-loop recycling of polyethylene terephthalate (PET). The polyester hydrolase PHL7, isolated from a compost metagenome, completely hydrolyzes amorphous PET films, releasing 91 mg of terephthalic acid per hour and mg of enzyme. Vertical scanning interferometry shows degradation rates of the PET film of 6.8 µm h-1 . Structural analysis indicates the importance of leucine at position 210 for the extraordinarily high PET-hydrolyzing activity of PHL7. Within 24 h, 0.6 mgenzyme gPET -1 completely degrades post-consumer thermoform PET packaging in an aqueous buffer at 70 °C without any energy-intensive pretreatments. Terephthalic acid recovered from the enzymatic hydrolysate is then used to synthesize virgin PET, demonstrating the potential of polyester hydrolases as catalysts in sustainable PET recycling processes with a low carbon footprint.

Reactive Sputtered Silicon Nitride as an Alternative Passivation Layer for Microelectrode Arrays in Sensitive Bioimpedimetric Cell Monitoring.

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.

Adhesion of Neurons and Glial Cells with Nanocolumnar TiN Films for Brain-Machine Interfaces.

Coupling of cells to biomaterials is a prerequisite for most biomedical applications; e.g., neuroelectrodes can only stimulate brain tissue in vivo if the electric signal is transferred to neurons attached to the electrodes' surface. Besides, cell survival in vitro also depends on the interaction of cells with the underlying substrate materials; in vitro assays such as multielectrode arrays determine cellular behavior by electrical coupling to the adherent cells. In our study, we investigated the interaction of neurons and glial cells with different electrode materials such as TiN and nanocolumnar TiN surfaces in contrast to gold and ITO substrates. Employing single-cell force spectroscopy, we quantified short-term interaction forces between neuron-like cells (SH-SY5Y cells) and glial cells (U-87 MG cells) for the different materials and contact times. Additionally, results were compared to the spreading dynamics of cells for different culture times as a function of the underlying substrate. The adhesion behavior of glial cells was almost independent of the biomaterial and the maximum growth areas were already seen after one day; however, adhesion dynamics of neurons relied on culture material and time. Neurons spread much better on TiN and nanocolumnar TiN and also formed more neurites after three days in culture. Our designed nanocolumnar TiN offers the possibility for building miniaturized microelectrode arrays for impedance spectroscopy without losing detection sensitivity due to a lowered self-impedance of the electrode. Hence, our results show that this biomaterial promotes adhesion and spreading of neurons and glial cells, which are important for many biomedical applications in vitro and in vivo.

Electrochemical Microwell Plate to Study Electroactive Microorganisms in Parallel and Real-Time.

Microbial resource mining of electroactive microorganism (EAM) is currently methodically hampered due to unavailable electrochemical screening tools. Here, we introduce an electrochemical microwell plate (ec-MP) composed of a 96 electrochemical deepwell plate and a recently developed 96-channel multipotentiostat. Using the ec-MP we investigated the electrochemical and metabolic properties of the EAM models Shewanella oneidensis and Geobacter sulfurreducens with acetate and lactate as electron donor combined with an individual genetic analysis of each well. Electrochemical cultivation of pure cultures achieved maximum current densities (j max) and coulombic efficiencies (CE) that were well in line with literature data. The co-cultivation of S. oneidensis and G. sulfurreducens led to an increased current density of j max of 88.57 ± 14.04 µA cm-2 (lactate) and j max of 99.36 ± 19.12 µA cm-2 (lactate and acetate). Further, a decreased time period of reaching j max and biphasic current production was revealed and the microbial electrochemical performance could be linked to the shift in the relative abundance.

Proliferation and Cluster Analysis of Neurons and Glial Cell Organization on Nanocolumnar TiN Sub-Strates.

Biomaterials employed for neural stimulation, as well as brain/machine interfaces, offer great perspectives to combat neurodegenerative diseases, while application of lab-on-a-chip devices such as multielectrode arrays is a promising alternative to assess neural function in vitro. For bioelectronic monitoring, nanostructured microelectrodes are required, which exhibit an increased surface area where the detection sensitivity is not reduced by the self-impedance of the electrode. In our study, we investigated the interaction of neurons (SH-SY5Y) and glial cells (U-87 MG) with nanocolumnar titanium nitride (TiN) electrode materials in comparison to TiN with larger surface grains, gold, and indium tin oxide (ITO) substrates. Glial cells showed an enhanced proliferation on TiN materials; however, these cells spread evenly distributed over all the substrate surfaces. By contrast, neurons proliferated fastest on nanocolumnar TiN and formed large cell agglomerations. We implemented a radial autocorrelation function of cellular positions combined with various clustering algorithms. These combined analyses allowed us to quantify the largest cluster on nanocolumnar TiN; however, on ITO and gold, neurons spread more homogeneously across the substrates. As SH-SY5Y cells tend to grow in clusters under physiologic conditions, our study proves nanocolumnar TiN as a potential bioactive material candidate for the application of microelectrodes in contact with neurons. To this end, the employed K-means clustering algorithm together with radial autocorrelation analysis is a valuable tool to quantify cell-surface interaction and cell organization to evaluate biomaterials' performance in vitro.

Advanced 96-microtiter plate based bioelectrochemical platform reveals molecular short cut of electron flow in cytochrome P450 enzyme.

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.

Electrochemical live monitoring of tumor cell migration out of micro-tumors on an innovative multiwell high-dense microelectrode array.

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.

Induced cross-resistance of BRAFV600E melanoma cells to standard chemotherapeutic dacarbazine after chronic PLX4032 treatment.

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 BRAFV600 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 BRAFV600E 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. BRAFV600E 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 BRAFV600E 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.

FEM-based design of optical transparent indium tin oxide multielectrode arrays for multiparametric, high sensitive cell based assays.

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.

A multidimensional impedance platform for the real-time analysis of single and combination drug pharmacology in patient-derived viable melanoma models.

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 BRAFV600E 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.

Comprehensive human stem cell differentiation in a 2D and 3D mode to cardiomyocytes for long-term cultivation and multiparametric monitoring on a multimodal microelectrode array setup.

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.