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.

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 cell-based impedance assay for monitoring transient receptor potential (TRP) ion channel activity.

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.

Immunocytochemical analysis of glycogen phosphorylase isozymes in the developing and adult retina of the domestic chicken (Gallus domesticus).

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.

Cells on a chip--the use of electric properties for highly sensitive monitoring of blood-derived factors involved in angiotensin II type 1 receptor signalling.

BACKGROUND: We developed a highly sensitive cardiomyocyte based screening system for the non-destructive electronic detection of chronotropic drugs and tissue-secreted factors involved in AT1 receptor-mediated cardiovascular diseases. METHODS: For this purpose we cultured spontaneously beating neonatal rat cardiomyocytes on microelectrode arrays (MEAs), and tested the optimised, stable culture parameters for a reproducible real-time recording of alterations in contraction frequency. After the evaluation of culture parameters, computer-based electronic measurement systems were used for counting of contractions by recording of the field potential of cardiomyocytes. RESULTS: Using the biosensor, angiotensin II, the predominant ligand of the AT1 receptor, was detected at very low concentrations of 10(-11) M via altered contractions of cardiomyocytes. Moreover, we demonstrated that cardiomyocyte coupled microarrays allow the detection of blood-derived low concentrated anti-AT1 receptor autoimmune antibodies of pregnant women suffering from preeclampsia. CONCLUSION: This study demonstrates the first well-suited electrophysiological recording of cardiomyocytes on multielectrode arrays as a benefit for functional biomonitoring for the detection of AT1 receptor/ligand interactions and other marker proteins in sera directed to cardiovascular diseases.

Artificial design of three-dimensional retina-like tissue from dissociated cells of the mammalian retina by rotation-mediated cell aggregation.

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.