In vitro cultured neuronal networks with defined connectivity are required to improve neuronal cell culture models. However, most protocols for their formation do not provide sufficient control of the direction and timing of neurite outgrowth with simultaneous access for analytical tools such as immunocytochemistry or patch-clamp recordings. Here, we present a proof-of-concept for the dynamic (i.e., time-gated) control of neurite outgrowth on a cell culture substrate based on 2D-micropatterned coatings of thermoresponsive polymers (TRP). The pattern consists of uncoated microstructures where neurons can readily adhere and neurites can extend along defined pathways. The surrounding regions are coated with TRP that does not facilitate cell or neurite growth at 33 °C. Increasing the ambient temperature to 37 °C renders the TRP coating cell adhesive and enables the crossing of gaps coated with TRP by neurites to contact neighboring cells. Here, we demonstrate the realization of this approach employing human neuronal SH-SY5Y cells and human induced neuronal cells. Our results suggest that this approach may help to establish a spatiotemporal control over the connectivity of multinodal neuronal networks.

Event Abstract Back to Event Switchable cell adhesive microstructures to grow defined neuronal networks Philipp Wysotzki1*, Jessica Schröder1, Laura V. Behm2, 3, Susanna Gerike2, 4, Felix Pfisterer2, Claus Duschl2, Michael Kirschbaum2, Jan Gimsa1 and Werner Baumann1 1 University of Rostock, Department of Biophysics, Germany 2 Fraunhofer-Institut für Zelltherapie und Immunologie (IZI), Branch Bioanalytics and Bioprocesses, Germany 3 Berlin-Brandenburgisches Zentrum für Regenerative Therapien, Charité Universitätsmedizin, Germany 4 Freie Universität Berlin, Germany The molecular structure of TRPs (thermo-responsive polymers) alters below and above a certain temperature. In experiments with neuronal cell lines (SH-SY5Y), we used TRP, which repelled cells below 33 °C and favored cell adhesion above 37 °C. For a more detailed characterization of the TRP properties, we determined the initial cell adhesion forces with single cell force spectroscopy. For the temporal and spatial control of the formation of neuronal circuits in vitro, micro-patterned structures with thermo-responsive surfaces were developed. Glass chips were produced with heating micro-structures covered by a Si3N4 passivation layer and coated with gold (Figure 1, gold). Experiments with primary neuronal mouse cells, which did not favor direct adhesion to TRP surfaces required micro-structuration of the TRP surfaces. Thiol-gold chemistry was used to coat the chip surfaces with TRP. The gold surface featured micro-structured round spots with protruding trenches, which prevented TRP adsorption but permitted the adhesion of single cells directly at the Si3N4 passivation layer (Figure 1, blue). The trenches, which connected the cell adhesion spots served to guide the axons of the cells to their neighbors (Figure 1, green). Cell adhesion to the feeder areas, trenches and cell adhesion spots could be enhanced by their specific surface modification with (3-aminopropyl)-triethoxysilane. The cell spots were connected to feeder areas on both sides of the chips (Figure 1, mint green). A gap in the trench to each neighboring cell prevented the direct connection of the axons to the neighbor cells (Figure 1, red). Heating micro-structures were located underneath the gaps. They permitting to switch between the cell adhesive and non-adhesive states of the TRP cover layer. Our final goal is the cultivation of primary neurons in defined network structures and the control of the connection in cellular arrays. Figure 1: Micro-structured chip for an array of four cells with two heating structures, four cell adhesion spot, and guiding trenches (blue: single cell adhesion spot, green: axon guide, red: thermo-switchable gap, gold: TRP-coated gold, mint green: feeder area). Scale bar: 100 µm. Figure 1 Acknowledgements The authors are grateful to the DFG (German Research Council) for funding the Projekt "NeuroTRP" (#290023374) Keywords: Thermo-responsive polymers, neuronal circuits, primary neuronal cells, Directed growth, microsystems Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Neural Networks Citation: Wysotzki P, Schröder J, Behm LV, Gerike S, Pfisterer F, Duschl C, Kirschbaum M, Gimsa J and Baumann W (2019). Switchable cell adhesive microstructures to grow defined neuronal networks. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00102 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 15 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Mr. Philipp Wysotzki, University of Rostock, Department of Biophysics, Rostock, Germany, 18057, Germany, Philipp.wysotzki@uni-rostock.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Philipp Wysotzki Jessica Schröder Laura V Behm Susanna Gerike Felix Pfisterer Claus Duschl Michael Kirschbaum Jan Gimsa Werner Baumann Google Philipp Wysotzki Jessica Schröder Laura V Behm Susanna Gerike Felix Pfisterer Claus Duschl Michael Kirschbaum Jan Gimsa Werner Baumann Google Scholar Philipp Wysotzki Jessica Schröder Laura V Behm Susanna Gerike Felix Pfisterer Claus Duschl Michael Kirschbaum Jan Gimsa Werner Baumann PubMed Philipp Wysotzki Jessica Schröder Laura V Behm Susanna Gerike Felix Pfisterer Claus Duschl Michael Kirschbaum Jan Gimsa Werner Baumann Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

We investigated the effects of alkaline pH on developing osteoblasts. Cells of the osteoblast-like cell line MC3T3-E1 were initially cultured for six days in HEPES-buffered media with pH ranging from 7.2 to 9.0. Cell count, cellular WST-1 metabolism, and ATP content were analyzed. The three parameters showed a pH optimum around pH 8.4, exceeding the recommended buffer range of HEPES at the alkaline flank. Therefore, only pH 7.2, 7.4, 7.8, and 8.4 media were used in more elaborate, daily investigations to reduce the effects of pH change within the pH control intervals of 24h. All parameters exhibited similar pH behaviors, roughly showing increases to 130% and 230% at pH 7.8 and 8.4, as well as decreases to 70% at pH 7.2 when using the pH 7.4 data for reference. To characterize cell differentiation and osteoblastic cell function, cells were cultured at pH 7.4 and under alkaline conditions at pH 7.8 and 8.4 for 14 days. Gene expression and mineralization were evaluated using microarray technology and Alizarin staining. Under alkaline conditions, ATF4, a regulator for terminal differentiation and function as well as DMP1, a potential marker for the transition of osteoblasts into osteocytes, were significantly upregulated, hinting at an accelerated differentiation process. After 21 days, significant mineralization was only detected at alkaline pH. We conclude that elevated pH is beneficial for the cultivation of bone cells and may also provide therapeutic value in bone regeneration therapies.

The effects of alkaline pH on the initial adhesion of osteoblasts to titanium surfaces was analyzed by single cell force microscopy (SCFM). In the SCFM measurements, the same cells were used to compare their unspecific adhesion to uncoated titanium with their specific adhesion to collagen coated titanium. When the maximum detachment forces (MDFs) were compared at pH 7.4 and 8.0, only slight differences were found on pure titanium, while the MDFs were significantly increased at collagen coated surfaces at pH 8.0. Effects on the subsequent proliferation and gene expression were investigated in an in vitro model system consisting of an alkalizing polyvinyl alcohol (PVA) matrix and a perforated titanium disc. The sodium hydroxide releasing matrix maintained the medium pH between pH 7.6 and pH 8.4 during the entire experiment. Under these conditions, cell counts were significantly increased with respect to the control system after 7 days in culture. These results were supported by gene expression analyses, which showed an upregulation of proliferation-controlling genes of the EGFR1 and PI3K/AKT pathways after 14 days in culture. The SCFM data were complemented by findings of an intensive regulation of genes known to be associated with focal adhesion such as Itga8 and Tnn.

We developed different types of glass cell-culture chips (GC3s) for culturing cells for microscopic observation in open media-containing troughs or in microfluidic structures. Platinum sensor and manipulation structures were used to monitor physiological parameters and to allocate and permeabilize cells. Electro-thermal micro pumps distributed chemical compounds in the microfluidic systems. The integrated temperature sensors showed a linear, Pt1000-like behavior. Cell adhesion and proliferation were monitored using interdigitated electrode structures (IDESs). The cell-doubling times of primary murine embryonic neuronal cells (PNCs) were determined based on the IDES capacitance-peak shifts. The electrical activity of PNC networks was detected using multi-electrode arrays (MEAs). During seeding, the cells were dielectrophoretically allocated to individual MEAs to improve network structures. MEA pads with diameters of 15, 20, 25, and 35 µm were tested. After 3 weeks, the magnitudes of the determined action potentials were highest for pads of 25 µm in diameter and did not differ when the inter-pad distances were 100 or 170 µm. Using 25-µm diameter circular oxygen electrodes, the signal currents in the cell-culture media were found to range from approximately −0.08 nA (0% O2) to −2.35 nA (21% O2). It was observed that 60-nm thick silicon nitride-sensor layers were stable potentiometric pH sensors under cell-culture conditions for periods of days. Their sensitivity between pH 5 and 9 was as high as 45 mV per pH step. We concluded that sensorized GC3s are potential animal replacement systems for purposes such as toxicity pre-screening. For example, the effect of mefloquine, a medication used to treat malaria, on the electrical activity of neuronal cells was determined in this study using a GC3 system.

We combined a multi-sensor glass-chip with a microfluidic channel grid for the characterization of cellular behavior. The grid was imprinted in poly-dimethyl-siloxane. Mouse-embryonal/fetal calvaria fibroblasts (MC3T3-E1) were used as a model system. Thin-film platinum (Pt) sensors for respiration (amperometric oxygen electrode), acidification (potentiometric pH electrodes) and cell adhesion (interdigitated-electrodes structures, IDES) allowed us to monitor cell-physiological parameters as well as the cell-spreading behavior. Two on-chip electro-thermal micro-pumps (ETμPs) permitted the induction of medium flow in the system, e.g., for medium mixing and drug delivery. The glass-wafer technology ensured the microscopic observability of the on-chip cell culture. Connecting Pt structures were passivated by a 1.2 μm layer of silicon nitride (Si3N4). Thin Si3N4 layers (20 nm or 60 nm) were used as the sensitive material of the pH electrodes. These electrodes showed a linear behavior in the pH range from 4 to 9, with a sensitivity of up to 39 mV per pH step. The oxygen sensors were circular Pt electrodes with a sensor area of 78.5 μm2. Their sensitivity was 100 pA per 1% oxygen increase in the range from 0% to 21% oxygen (air saturated). Two different IDES geometries with 30- and 50-μm finger spacings showed comparable sensitivities in detecting the proliferation rate of MC3T3 cells. These cells were cultured for 11 days in vitro to test the biocompatibility, microfluidics and electric sensors of our system under standard laboratory conditions.

We developed a confined microfluidic cell culture system with a bottom plate made of a microscopic slide with planar platinum sensors for the measurement of acidification, oxygen consumption, and cell adhesion. The slides were commercial slides with indium tin oxide (ITO) plating or were prepared from platinum sputtering (100 nm) onto a 10-nm titanium adhesion layer. Direct processing of the sensor structures (approximately three minutes per chip) by an ultrashort pulse laser facilitated the production of the prototypes. pH-sensitive areas were produced by the sputtering of 60-nm Si3N4 through a simple mask made from a circuit board material. The system body and polydimethylsiloxane (PDMS) molding forms for the microfluidic structures were manufactured by micromilling using a printed circuit board (PCB) milling machine for circuit boards. The microfluidic structure was finally imprinted in PDMS. Our approach avoided the use of photolithographic techniques and enabled fast and cost-efficient prototyping of the systems. Alternatively, the direct production of metallic, ceramic or polymeric molding tools was tested. The use of ultrashort pulse lasers improved the precision of the structures and avoided any contact of the final structures with toxic chemicals and possible adverse effects for the cell culture in lab-on-a-chip systems.

Beim aktuellen Entwicklungsstand unseres PoreGenic ® Patch-on-Chip-Systems wurden für Signalableitungen von adhärenten Zellen die pipettenähnlichen Patch-Sites auf 10 µm verlängert und eine Zellpositionierung etabliert.Nach unserem Kenntnisstand ist es uns damit weltweit erstmalig gelungen, adhärente Säugetierzellen mit einem automatisierbaren Patch-on

Automated patch-clamp setups are applied to investigate dose response relationships and target kinetics in the development of new pharmaceutical agents. Currently, automated systems are limited to investigations of suspended single cells. We pursue the development of assays for detecting the membrane properties in adherent networks because the majority of cells in humans grow adherently. In a first step, we developed PoreGenic®, a novel patch-clamp system for cells growing on a sensor chip with micro-structured needle electrodes arranged in an 8×8 multi-electrode array with a pitch of 100 μm. PoreGenic® allows for the electrical cell manipulation as well as for extra- and intracellular potential measurements. Four types of needle electrodes of different shapes and materials were tested with heights of less than 10 μm. For intracellular detection, electroporation pulses were applied to form membrane pores for the introduction of the electrodes into the cytoplasm. Fluorescence and scanning-electron microscopy in combination with focused ion beam preparation were used to characterize the success of electroporation. In a number of experiments, we could access the cytoplasm and detect intracellular potentials. Our current system features 16 hollow needle structures with fluidic connections for patch-clamp experiments.

Summary We report the investigation of the interfaces between microneedle arrays and cell cultures in patch‐on‐chip systems by using Focused Ion Beam (FIB) preparation and Scanning Electron Microscopy (SEM). First, FIB preparations of micro chips are made to determine the size and shape of the designed microneedles. In this essay, we investigate the cell‐substrate interaction, especially the cell adhesion, and the microneedle's potential cell penetration. For this purpose, cross‐sectional preparation of these hard/soft hybrid structures is performed by the FIB technology. By applying the FIB technology followed by high‐resolution imaging with SEM, new insights into the cell‐substrate interface can be received. One can clearly distinguish between cells that are only in contact with microneedles and cells that are penetrated by microneedles. A stack of slice images is collected by the application of the slice‐and‐view setup during FIB preparation and is used for threedimensional reconstruction of cells and micro‐needles. SCANNING 34: 221‐229, 2012. © 2011 Wiley Periodicals, Inc.

We present a label-free in vitro method for testing the toxic potentials of chemical substances using primary neuronal cells. The cells were prepared from 16-day-old NMRI mouse embryos and cultured on silicon chips (www.bionas.de) under the influence of different parathion concentrations with sensors for respiration (Clark-type oxygen electrodes), acidification (pH-ISFETs) and cell adhesion (interdigitated electrode structures, IDES). After 12 days in vitro, the sensor readouts were simultaneously recorded for 350 min in the presence of parathion applying a serial 1:3 dilution. The parathion-dependent data was fitted by logistic functions. IC(50) values of approximately 105 μM, 65 μM, and 54 μM were found for respiration, acidification, and adhesion, respectively. An IC(50) value of approximately 36 μM was determined from the intracellular ATP-levels of cells, which were detected by an ATP-luminescence assay using micro-well plates. While the intracellular ATP level and cell adhesion showed no deviation from a simple logistic decay, increases of approximately 29% in the respiration and 15% in the acidification rates above the control values were found at low parathion concentrations, indicating hormesis. These increases could be fitted by a modified logistic function. We believe that the label-free, continuous, multi-parametric monitoring of cell-metabolic processes may have applications in systems-biology and biomedical research, as well as in environmental monitoring. The parallel characterization of IC(50) values and hormetic effects may provide new insights into the metabolic mechanisms of toxic challenges to the cell.

We developed a modular neurochip system by combining a small (16 x 16 mm(2)) glass neurochip (GNC) with a homemade head stage and commercial data acquisition hardware and software. The system is designed for the detection of the electric activity of cultivated nerve or muscle cells by a 52-microelectrode array (MEA). In parallel, cell adhesion can be registered from the electric impedance of an interdigitated electrode structure (IDES). The GNC was tested with various cell lines and primary cells. It is fully autoclavable and re-useable. Murine embryonic primary cells were used as a model system to correlate the electric activity and adhesion of neuronal networks in a drug test with sodium valproic acid. The test showed the advantage of the parallel IDES and MEA measurements, i.e. the parallel detection of cytotoxic and neurotoxic effects. Toxic exposure of the cells during neuronal network formation allows for the characterization of developmental neurotoxic effects even at drug concentrations below the EC(50)-value for acute neurotoxic effects. At high drug concentrations, the degree of cytotoxic damage can still be assessed from the IDES data in the event that no electric activity develops. The GNC provides optimal cell culture conditions for up to months in combination with full microscopic observability. The 4 '' glass wafer technology allows for a high precision of the GNC structures and an economic production of our new system that can be applied in general and developmental toxicity tests as well as in the search for neuro-active compounds.

This paper reports on the fabrication of hollow microneedle electrodes with fluidic channels arranged in 8times8 arrays. Features of these electrodes include (i) an increased surface area for improved intracellular potential measurements with simultaneous membrane cell poration capabilities, (ii) their potential use in highly parallel patch-clamp applications and (iii) the ability to efficiently inject reagents and extract cytoplasm into and from the cell interior, respectively. Three different fabrication processes to realize hollow microneedle electrode arrays with incorporated microfluidic components, as well as initial experiments with cell cultures, are presented.

We present a new sensor chip system for intracellular potential measurements of adherently growing cells using micro-structured needle electrode (MNE) arrays. Existing methods for intracellular investigations are time-consuming, tedious or limited to the analysis of suspended cells. However, most biological cells grow adherently. To overcome these methodological limitations a novel technique, local micro-invasive needle electroporation (LOMINE) in MNE arrays, has been developed. LOMINE opens the cell membrane for introducing a MNE into the cytoplasm. This paper describes the fabrication process of the MNE-array chips and first cell electroporation experiments.