A flexible implant for acute intrapancreatic electrophysiology.

Microelectrode arrays (MEAs) have proven to be a powerful tool to study electrophysiological processes over the last decades with most technology developed for investigation of the heart or brain. Other targets in the field of bioelectronic medicine are the peripheral nervous system and its innervation of various organs. Beyond the heart and nervous systems, the beta cells of the pancreatic islets of Langerhans generate action potentials during the production of insulin. In vitro experiments have demonstrated that their activity is a biomarker for blood glucose levels, suggesting that recording their activity in vivo could support patients suffering from diabetes mellitus with long-term automated read-out of blood glucose concentrations. Here, we present a flexible polymer-based implant having 64 low impedance microelectrodes designed to be implanted to a depth of 10 mm into the pancreas. As a first step, the implant will be used in acute experiments in pigs to explore the electrophysiological processes of the pancreas in vivo. Beyond use in the pancreas, our flexible implant and simple implantation method may also be used in other organs such as the brain.

Proteomic responses in the human dopaminergic LUHMES cell line to imidacloprid and its metabolites imidacloprid-olefin and desnitro-imidacloprid.

Neonicotinoids (neonics) are amongst the most commonly used class of pesticides globally. In the United States, imidacloprid (IMI) is extensively used for agriculture and in other common applications such as house-hold pest control. Regular exposure to IMI, and several of its known metabolites including IMI-olefin and desnitro-imidacloprid (DN-IMI), has been shown to be harmful to many organisms including mammals, birds, and fish. Studies show that neonics bind human nicotinicacetylcholine receptors (nAChRs) and cause cellular toxicity. In the dopaminergic Lund human mesencephalic (LUHMES) cell line, IMI and other neonics (10-100 µM) have been recently shown to activate intracellular calcium signaling through nAChRs. Thus, we examined proteomic responses of LUHMES cells to a 48-h treatment with 50 µM IMI, IMI-olefin, or DN-IMI. Our findings show differential effects of these neonics on cellular protein expression. Bioinformatic analysis of significantly altered proteins indicates an effect of IMI, IMI-olefin, and DN-IMI on protein synthesis and ribosomal function. These findings suggest a role for protein synthesis and transcriptional regulation in neonic-mediated dopaminergic neurotoxicity.

Human neuronal signaling and communication assays to assess functional neurotoxicity.

Prediction of drug toxicity on the human nervous system still relies mainly on animal experiments. Here, we developed an alternative system allowing assessment of complex signaling in both individual human neurons and on the network level. The LUHMES cultures used for our approach can be cultured in 384-well plates with high reproducibility. We established here high-throughput quantification of free intracellular Ca2+ concentrations [Ca2+]i as broadly applicable surrogate of neuronal activity and verified the main processes by patch clamp recordings. Initially, we characterized the expression pattern of many neuronal signaling components and selected the purinergic receptors to demonstrate the applicability of the [Ca2+]i signals for quantitative characterization of agonist and antagonist responses on classical ionotropic neurotransmitter receptors. This included receptor sub-typing and the characterization of the anti-parasitic drug suramin as modulator of the cellular response to ATP. To exemplify potential studies on ion channels, we characterized voltage-gated sodium channels and their inhibition by tetrodotoxin, saxitoxin and lidocaine, as well as their opening by the plant alkaloid veratridine and the food-relevant marine biotoxin ciguatoxin. Even broader applicability of [Ca2+]i quantification as an end point was demonstrated by measurements of dopamine transporter activity based on the membrane potential-changing activity of this neurotransmitter carrier. The substrates dopamine or amphetamine triggered [Ca2+]i oscillations that were synchronized over the entire culture dish. We identified compounds that modified these oscillations by interfering with various ion channels. Thus, this new test system allows multiple types of neuronal signaling, within and between cells, to be assessed, quantified and characterized for their potential disturbance.

Acute effects of the imidacloprid metabolite desnitro-imidacloprid on human nACh receptors relevant for neuronal signaling.

Several neonicotinoids have recently been shown to activate the nicotinic acetylcholine receptor (nAChR) on human neurons. Moreover, imidacloprid (IMI) and other members of this pesticide family form a set of diverse metabolites within crops. Among these, desnitro-imidacloprid (DN-IMI) is of special toxicological interest, as there is evidence (i) for human dietary exposure to this metabolite, (ii) and that DN-IMI is a strong trigger of mammalian nicotinic responses. We set out here to quantify responses of human nAChRs to DN-IMI and an alternative metabolite, IMI-olefin. To evaluate toxicological hazards, these data were then compared to those of IMI and nicotine. Ca2+-imaging experiments on human neurons showed that DN-IMI exhibits an agonistic effect on nAChRs at sub-micromolar concentrations (equipotent with nicotine) while IMI-olefin activated the receptors less potently (in a similar range as IMI). Direct experimental data on the interaction with defined receptor subtypes were obtained by heterologous expression of various human nAChR subtypes in Xenopus laevis oocytes and measurement of the transmembrane currents evoked by exposure to putative ligands. DN-IMI acted on the physiologically important human nAChR subtypes α7, α3ß4, and α4ß2 (high-sensitivity variant) with similar potency as nicotine. IMI and IMI-olefin were confirmed as nAChR agonists, although with 2-3 orders of magnitude lower potency. Molecular docking studies, using receptor models for the α7 and α4ß2 nAChR subtypes supported an activity of DN-IMI similar to that of nicotine. In summary, these data suggest that DN-IMI functionally affects human neurons similar to the well-established neurotoxicant nicotine by triggering α7 and several non-α7 nAChRs.

Functional alterations by a subgroup of neonicotinoid pesticides in human dopaminergic neurons.

Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca2+ concentration ([Ca2+]i) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of α7 and non-α7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca2+]i signaling at 10-100 µM. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the α7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1-10 µM) blunted the signaling response of nicotine. The pesticides (at 3-30 µM) also blunted the response to the non-α7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.

Correlative Microscopy Combining Secondary Ion Mass Spectrometry and Electron Microscopy: Comparison of Intensity-Hue-Saturation and Laplacian Pyramid Methods for Image Fusion.

Correlative microscopy combining various imaging modalities offers powerful insights into obtaining a comprehensive understanding of physical, chemical, and biological phenomena. In this article, we investigate two approaches for image fusion in the context of combining the inherently lower-resolution chemical images obtained using secondary ion mass spectrometry (SIMS) with the high-resolution ultrastructural images obtained using electron microscopy (EM). We evaluate the image fusion methods with three different case studies selected to broadly represent the typical samples in life science research: (i) histology (unlabeled tissue), (ii) nanotoxicology, and (iii) metabolism (isotopically labeled tissue). We show that the intensity-hue-saturation fusion method often applied for EM-sharpening can result in serious image artifacts, especially in cases where different contrast mechanisms interplay. Here, we introduce and demonstrate Laplacian pyramid fusion as a powerful and more robust alternative method for image fusion. Both physical and technical aspects of correlative image overlay and image fusion specific to SIMS-based correlative microscopy are discussed in detail alongside the advantages, limitations, and the potential artifacts. Quantitative metrics to evaluate the results of image fusion are also discussed.

Cell Therapy by Mesenchymal Stromal Cells Versus Myoblasts in a Pig Model of Urinary Incontinence.

The leading cause of stress urinary incontinence (SUI) in women is the urethral sphincter muscle deficiency caused by mechanical stress during pregnancy and vaginal delivery. In men, prostate cancer surgery and injury of local nerves and muscles are associated with incontinence. Current treatment often fails to satisfy the patient's needs. Cell therapy may improve the situation. We therefore investigated the regeneration potential of cells in ameliorating sphincter muscle deficiency and UI in a large animal model. Urethral sphincter deficiency was induced surgically in gilts by electrocautery and balloon dilatation. Adipose tissue-derived stromal cells (ADSCs) and myoblasts from Musculus semitendinosus were isolated from male littermates, expanded, characterized in depth for expression of marker genes and in vitro differentiation, and labeled. The cells were injected into the deficient sphincter complex of the incontinent female littermates. Incontinent gilts receiving no cell therapy served as controls. Sphincter deficiency and functional regeneration were recorded by monitoring the urethral wall pressure during follow-up by two independent methods. Cells injected were detected in vivo during follow-up by transurethral fluorimetry, ex vivo by fluorescence imaging, and in cryosections of tissues targeted by immunofluorescence and by polymerase chain reaction of the sex-determining region Y (SRY) gene. Partial spontaneous regeneration of sphincter muscle function was recorded in control gilts, but the sphincter function remained significantly below levels measured before induction of incontinence (67.03% ± 14.00%, n = 6, p < 0.05). Injection of myoblasts yielded an improved sphincter regeneration within 5 weeks of follow-up but did not reach significance compared to control gilts (81.54% ± 25.40%, n = 5). A significant and full recovery of the urethral sphincter function was observed upon injection of ADSCs within 5 weeks of follow-up (100.4% ± 23.13%, n = 6, p < 0.05). Injection of stromal cells provoked slightly stronger infiltration of CD45pos leukocytes compared to myoblasts injections and controls. The data of this exploratory study indicate that ADSCs inherit a significant potential to regenerate the function of the urethral sphincter muscle.

Elements and development processes for test methods in toxicology and human health-relevant life science research.

Many laboratory procedures generate data on properties of chemicals, but they cannot be equated with toxicological "test methods". This apparent discrepancy is not limited to in vitro testing, using animal-free new approach methods (NAM), but also applies to animal-based testing approaches. Here, we give a brief overview of the differences between data generation and the setup or use of a complete test method. While there is excellent literature available on this topic for specialists (GIVIMP guidance; ToxTemp overview), a brief overview and easily-accessible entry point may be useful for a broader community. We provide a single figure to summarize all test method elements and processes required in the development (setup and adaptation) of a test method. The exposure scheme, the endpoint, and the test system are briefly outlined as fundamental elements of any test method. A rationale is provided, why they are not sufficient. We then explain the importance and role of purpose definition (including some information on what is modelled) and the prediction model, aka data interpretation procedure, which depends on the purpose definition, as further essential elements. This connection exemplifies that all fundamental elements are interdependent, and none can be omitted. Finally, discussion is provided on validation as a measure to provide confidence in the reliability, performance, and relevance of a test method. In this sense, validation may be considered a sixth fundamental element for practical use of test methods.

Many laboratory procedures generate data on chemicals, but they cannot be considered complete toxicological "test methods". Here, we give a brief explanation of the fundamental elements of a toxicological test method. We provide an illustration that gives a complete overview of the devel­opment of a test method for non-specialists. We introduce the six fundamental elements, i.e., the exposure scheme, the test endpoint, the test system, the purpose definition and the prediction model and describe how they work together. Finally, we discuss the concept of validation. An understanding of these concepts is important for good-quality scientific research and especially for the development and acceptance of alternatives to animal experiments.

Cell-based therapy for the deficient urinary sphincter.

When sterile culture techniques of mammalian cells first became state of the art, there was tremendous anticipation that such cells could be eventually applied for therapeutic purposes. The discovery of adult human stem or progenitor cells further motivated scientists to pursue research in cell-based therapies. Although evidence from animal studies suggests that application of cells yields measurable benefits, in urology and many other disciplines, progenitor-cell-based therapies are not yet routinely clinically available. Stress urinary incontinence (SUI) is a condition affecting a large number of patients. The etiology of SUI includes, but is not limited to, degeneration of the urinary sphincter muscle tissue and loss of innervation, as well as anatomical and biomechanical causes. Therefore, different regimens were developed to treat SUI. However, at present, a curative functional treatment is not at hand. A progenitor-cell-based therapy that can tackle the etiology of incontinence, rather than the consequences, is a promising strategy. Therefore, several research teams have intensified their efforts to develop such a therapy for incontinence. Here, we introduce candidate stem and progenitor cells suitable for SUI treatment, show how the functional homogeneity and state of maturity of differentiated cells crucial for proper tissue integration can be assessed electrophysiologically prior to their clinical application, and discuss the trophic potential of adult mesenchymal stromal (or stem) cells in regeneration of neuronal function.

Assay for evaluation of proarrhythmic effects of herbal products: Case study with 12 Evodia preparations.

Guidelines for preclinical drug development reduce the occurrence of arrhythmia-related side effects. Besides ample evidence for the presence of arrhythmogenic substances in plants, there is no consensus on a research strategy for the evaluation of proarrhythmic effects of herbal products. Here, we propose a cardiac safety assay for the detection of proarrhythmic effects of plant extracts based on the experimental approaches described in the Comprehensive In vitro Proarrhythmia Assay (CiPA). Microelectrode array studies (MEAs) and voltage sensing optical technique on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were combined with ionic current measurements in mammalian cell lines, In-silico simulations of cardiac action potentials (APs) and statistic regression analysis. Proarrhythmic effects of 12 Evodia preparations, containing different amounts of the hERG inhibitors dehydroevodiamine (DHE) and hortiamine were analysed. Extracts produced different prolongation of the AP, occurrence of early after depolarisations and triangulation of the AP in hiPSC-CMs depending on the contents of the hERG inhibitors. DHE and hortiamine dose-dependently prolonged the field potential duration in hiPSC-CMs studied with MEAs. In-silico simulations of ventricular AP support a scenario where proarrhythmic effects of Evodia extracts are predominantly caused by the content of the selective hERG inhibitors. Statistic regression analysis revealed a high torsadogenic risk for both compounds that was comparable to drugs assigned to the high-risk category in a CiPA study.

Laser-Induced Action Potential-Like Measurements of Cardiomyocytes on Microelectrode Arrays for Increased Predictivity of Safety Pharmacology.

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small proarrhythmic membrane potential fluctuations. The shape and time course of the repolarizing fraction of the AP can be pivotal for the presence or absence of arrhythmia. Microelectrode arrays (MEA) allow easy access to cardiotoxic compound effects via extracellular field potentials (FP). Although a powerful and well-established tool in research and cardiac safety pharmacology, the FP waveform does not allow to infer the original AP shape due to the extracellular recording principle and the resulting intrinsic alternating current (AC) filtering. A novel device, described here, can repetitively open the membrane of cardiomyocytes cultivated on top of the MEA electrodes at multiple cultivation time points, using a highly focused nanosecond laser beam. The laser poration results in transforming the electrophysiological signal from FP to intracellular-like APs (laser-induced AP, liAP) and enables the recording of transcellular voltage deflections. This intracellular access allows a better description of the AP shape and a better and more sensitive classification of proarrhythmic potentials than regular MEA recordings. This system is a revolutionary extension to the existing electrophysiological methods, permitting accurate evaluation of cardiotoxic effect with all advantages of MEA-based recordings (easy, acute, and chronic experiments, signal propagation analysis, etc.).

Rapid functional evaluation of beta-cells by extracellular recording of membrane potential oscillations with microelectrode arrays.

The membrane potential (V (m)) of beta-cells oscillates at glucose concentrations between ~6 and 25 mM, i.e. burst phases with action potentials alternate with silent interburst phases generating so-called slow waves. The slow waves drive oscillations of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) and insulin secretion. The length of the bursts correlates with the amount of insulin release. Thus, the fraction of plateau phase (FOPP), i.e. the percentage of time with burst activity, is an excellent marker for beta-cell function and metabolic integrity. Extracellular voltage changes of mouse islets were measured using a microelectrode array (MEA) allowing the detection of burst and interburst phases. At a non-stimulating glucose concentration (3 mM) no electrical activity was detectable while bursting was continuous at 30 mM. The glucose concentration-response (determined as FOPP) curve revealed half-maximal stimulation at 12 ± 1 mM (Hill equation fit). The signal was sensitive to K(ATP) channel modulators, e.g. tolbutamide or diazoxide. Simultaneous recordings of electrical activity and [Ca(2+)](c) revealed congruent bursts and peaks, respectively. The extracellular recordings are in perfect agreement with more time-consuming intracellular electrical recordings. The results provide a 'proof-of-principle' for detection of beta-cell slow waves and determination of the FOPP using extracellular electrodes in a MEA-based system. The method is facile and provides the capability to study the effects of modulators of beta-cell function including possible anti-diabetic drugs in real time. Moreover, the method may be useful for checking the metabolic integrity of human donor islets prior to transplantation.

Cardiotoxicity testing using pluripotent stem cell-derived human cardiomyocytes and state-of-the-art bioanalytics: a review.

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art bioanalytical methods and sensors is reviewed. The focus is on hESC-derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given.

The potential of induced pluripotent stem cells for discriminating neurodevelopmental disorders.

Studying human disease-specific processes and mechanisms in vitro is limited by a lack of valid human test systems. Induced pluripotent stem cells (iPSCs) evolve as an important and promising tool to better understand the molecular pathology of neurodevelopmental disorders. Patient-derived iPSCs enable analysis of unique disease mechanisms and may also serve for preclinical drug development. Here, we review the current knowledge on iPSC models for schizophrenia and autism spectrum disorders with emphasis on the discrimination between them. It appears that transcriptomic analyses and functional read-outs are the most promising approaches to uncover specific disease mechanisms in vitro.

Incorporation of stem cell-derived astrocytes into neuronal organoids to allow neuro-glial interactions in toxicological studies.

Human cell-based neural organoids are increasingly being used for investigations of neurotoxicity, and to study the pathophysiology of neurodegenerative diseases. Here, we present a fast and robust method to generate 3D cultured human dopaminergic neurons (LUHMES) for toxicity testing and long-term culture. Moreover, a plating step was introduced to allow generation of neurite networks with defined 2D orientation and several mm length, while all cell bodies (somata) remained in a 3D, dome-like structure. These cultures, named here 2.5D (for 2.5 dimensional), offer new approaches to quantify toxicant effects on organoids by standard technology and high throughput. For instance, the system reacted to the parkinsonian model toxicants MPP+, rotenone, MG-132 and the ferroptosis-inducer erastin. Moreover, stable incorporation of human stem cell-derived astrocytes or microglia was possible. Added astrocytes stabilized the post mitotic state of the LUHMES neurons and thereby allowed the formation of a stable micro-physiological system. We observed neuroprotection against the proteasome inhibitor MG-132 and the ferroptosis-inducer erastin by such glia. This exemplifies the crucial protective role of astrocytes in neurodegeneration. The modularity of the system was further employed to incorporate microglia together with astrocytes into the organoids. Such ratio-defined, three cell type-based organoids will allow new approaches to study human pathophysiology and toxicology of the nervous system.

Non-invasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model.

The increasing prevalence of diabetes, its heterogeneity, and the limited number of treatment options drive the need for physiologically relevant assay platforms with human genetic background that have the potential to improve mechanistic understanding and e\xpedite diabetes-related research and treatment. In this study, we developed an endocrine pancreas-on-a-chip model based on a tailored microfluidic platform, which enables self-guided trapping of single human pseudo-islets. Continuous, low-shear perfusion provides a physiologically relevant microenvironment especially important for modeling and monitoring of the endocrine function as well as sufficient supply with nutrients and oxygen. Human pseudo-islets, generated from the conditionally immortalized EndoC-ßH3 cell line, were successfully injected by hydrostatic pressure-driven flow without altered viability. To track insulin secretion kinetics in response to glucose stimulation in a time-resolved manner, dynamic sampling of the supernatant as well as non-invasive real-time monitoring using Raman microspectroscopy was established on-chip. Dynamic sampling indicated a biphasic glucose-stimulated insulin response. Raman microspectroscopy allowed to trace glucose responsiveness in situ and to visualize different molecular structures such as lipids, mitochondria and nuclei. In-depth spectral analyses demonstrated a glucose stimulation-dependent, increased mitochondrial activity, and a switch in lipid composition of insulin secreting vesicles, supporting the high performance of our pancreas-on-a-chip model.

Inhibitory postsynaptic potentials carry synchronized frequency information in active cortical networks.

Temporal precision in spike timing is important in cortical function, interactions, and plasticity. We found that, during periods of recurrent network activity (UP states), cortical pyramidal cells in vivo and in vitro receive strong barrages of both excitatory and inhibitory postsynaptic potentials, with the inhibitory potentials showing much higher power at all frequencies above approximately 10 Hz and more synchrony between nearby neurons. Fast-spiking inhibitory interneurons discharged strongly in relation to higher-frequency oscillations in the field potential in vivo and possess membrane, synaptic, and action potential properties that are advantageous for transmission of higher-frequency activity. Intracellular injection of synaptic conductances having the characteristics of the recorded EPSPs and IPSPs reveal that IPSPs are important in controlling the timing and probability of action potential generation in pyramidal cells. Our results support the hypothesis that inhibitory networks are largely responsible for the dissemination of higher-frequency activity in cortex.

Assay Procedures for Compound Testing of hiPSC-Derived Cardiomyocytes Using Multiwell Microelectrode Arrays.

The cardiac action potential requires a precise timing of activation and inactivation of ion channel subtypes. Deviations, for example, due to blockage of specific voltage-gated potassium channels, can result in live-threatening arrhythmias. Due to the limitations of standard cellular assays based on cells which artificially express only single ion channel subtypes, many potentially interesting compounds are discarded during drug development. More predictive functional assays are required. With the upcoming of human stem-cell derived cardiomyocytes (hiPS-CM) these assays are available, supporting even the design of patient-derived disease models. Microelectrode array systems allow to noninvasively record and evaluate cardiac field action potentials. In this chapter we describe how to cultivate hiPS-CM on two parallelized MEA systems and suggest an experimental strategy for compound tests.

Comparative characterization of human induced pluripotent stem cells (hiPSC) derived from patients with schizophrenia and autism.

Human induced pluripotent stem cells (hiPSC) provide an attractive tool to study disease mechanisms of neurodevelopmental disorders such as schizophrenia. A pertinent problem is the development of hiPSC-based assays to discriminate schizophrenia (SZ) from autism spectrum disorder (ASD) models. Healthy control individuals as well as patients with SZ and ASD were examined by a panel of diagnostic tests. Subsequently, skin biopsies were taken for the generation, differentiation, and testing of hiPSC-derived neurons from all individuals. SZ and ASD neurons share a reduced capacity for cortical differentiation as shown by quantitative analysis of the synaptic marker PSD95 and neurite outgrowth. By contrast, pattern analysis of calcium signals turned out to discriminate among healthy control, schizophrenia, and autism samples. Schizophrenia neurons displayed decreased peak frequency accompanied by increased peak areas, while autism neurons showed a slight decrease in peak amplitudes. For further analysis of the schizophrenia phenotype, transcriptome analyses revealed a clear discrimination among schizophrenia, autism, and healthy controls based on differentially expressed genes. However, considerable differences were still evident among schizophrenia patients under inspection. For one individual with schizophrenia, expression analysis revealed deregulation of genes associated with the major histocompatibility complex class II (MHC class II) presentation pathway. Interestingly, antipsychotic treatment of healthy control neurons also increased MHC class II expression. In conclusion, transcriptome analysis combined with pattern analysis of calcium signals appeared as a tool to discriminate between SZ and ASD phenotypes in vitro.

A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes.

For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.