PEM Electrochemical Hydrogen Compression with Sputtered Pt Catalysts.

This work presents research on thin magnetron-sputtered platinum (Pt) films deposited over commercial gas diffusion electrodes and applied to convert and pressurize hydrogen in an electrochemical hydrogen pump. The electrodes were integrated into a membrane electrode assembly with a proton conductive membrane. Their electrocatalytic efficiency toward hydrogen oxidation and hydrogen evolution reactions was studied in a self-made laboratory test cell by means of steady-state polarization curves and cell voltage measurements (U/j and U/pdiff characteristics). The achieved current density at a cell voltage of 0.5 V, the atmospheric pressure of the input hydrogen, and a temperature of 60 °C was more than 1.3 A cm-2. The registered increase in the cell voltage with the increasing pressure was only 0.05 mV bar-1. Comparative data with commercial E-TEK electrodes reveal the superior catalyst performance and essential cost reduction of the electrochemical hydrogen conversion on the sputtered Pt films.

Viscoelastic Properties of Zona Pellucida of Oocytes Characterized by Transient Electrical Impedance Spectroscopy.

The success rate in vitro fertilization is significantly linked to the quality of the oocytes. The oocyte's membrane is encapsulated by a shell of gelatinous extracellular matrix, called zona pellucida, which undergoes dynamic changes throughout the reproduction cycle. During the window of highest fertility, the zona pellucida exhibits a softening phase, while it remains rigid during oocyte maturation and again after fertilization. These variations in mechanical properties facilitate or inhibit sperm penetration. Since successful fertilization considerably depends on the state of the zona pellucida, monitoring of the hardening process of the zona pellucida is vital. In this study, we scrutinized two distinct genetic mouse models, namely, fetuin-B wild-type and fetuin-B/ovastacin double deficient with normal and super-soft zona pellucida, respectively. We evaluated the hardening with the help of a microfluidic aspiration-assisted electrical impedance spectroscopy system. An oocyte was trapped by a microhole connected to a microfluidic channel by applying suction pressure. Transient electrical impedance spectra were taken by microelectrodes surrounding the microhole. The time-depending recovery of zona pellucida deflections to equilibrium was used to calculate the Young's modulus and, for the first time, absolute viscosity values. The values were obtained by fitting the curves with an equivalent mechanical circuit consisting of a network of dashpots and springs. The observer-independent electrical readout in combination with a fitting algorithm for the calculation of the viscoelastic properties demonstrates a step toward a more user-friendly and easy-to-use tool for the characterizing and better understanding of the rheological properties of oocytes.

The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates.

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 µm from the outer rim of iPSC colonies. Single-cell RNA-sequencing and spatial reconstruction of gene expression demonstrated that OCT4high subsets, residing at the edge of the colony, have pronounced up-regulation of the TGF-ß pathway, particularly of NODAL and its inhibitor LEFTY. Interestingly, after 5-7 days, iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size without enzymatic or mechanical treatment. Within the early 3D aggregates, radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Early self-detached aggregates revealed up-regulated germline-specific gene expression patterns as compared to conventional EBs. However, there were no marked differences after further directed differentiation toward hematopoietic, mesenchymal, and neuronal lineages. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

Dry Film Resist Laminated Microfluidic System for Electrical Impedance Measurements.

In micro-electrical-mechanical systems (MEMS), thick structures with high aspect ratios are often required. Dry film photoresist (DFR) in various thicknesses can be easily laminated and patterned using standard UV lithography. Here, we present a three-level DFR lamination process of SUEX for a microfluidic chip with embedded, vertically arranged microelectrodes for electrical impedance measurements. To trap and fix the object under test to the electrodes, an aperture is formed in the center of the ring-shaped electrodes in combination with a microfluidic suction channel underneath. In a proof-of-concept, the setup is characterized by electrical impedance measurements with polystyrene and ZrO2 spheres. The electrical impedance is most sensitive at approximately 2 kHz, and its magnitudes reveal around 200% higher values when a sphere is trapped. The magnitude values depend on the sizes of the spheres. Electrical equivalent circuits are applied to simulate the experimental results with a close match.

Electrochemical Impedance Spectroscopy Biosensor Enabling Kinetic Monitoring of Fucosyltransferase Activity.

Monitoring glycosyltransferases on biosensors is of great interest for pathogen and cancer diagnostics. As a proof of concept, we here demonstrate the layer-by-layer immobilization of a multivalent neoglycoprotein (NGP) as a substrate for a bacterial fucosyltransferase (FucT) and the subsequent binding of the fucose-specific Aleuria aurantia lectin (AAL) on an electrochemical impedance spectroscopy (EIS) sensor. We report for the first time the binding kinetics of a glycosyltransferase in real-time. Highly stable EIS measurements are obtained by the modification of counter and reference electrodes with polypyrrole: polystyrene sulfonate (PPy:PSS). In detail, the N-acetyllactosamine (LacNAc)-carrying NGP was covalently immobilized on the gold working electrode and served as a substrate for the FucT-catalyzed reaction. The LacNAc epitopes were converted to Lewisx (Lex) and detected by AAL. AAL binding to the Lex epitope was further confirmed in a lectin displacement and a competitive lectin binding inhibition experiment. We monitored the individual kinetic processes via EIS. The time constant for covalent immobilization of the NGP was 653 s. The FucT kinetics was the slowest process with a time constant of 1121 s. In contrast, a short time constant of 11.8 s was determined for the interaction of AAL with the modified NGPs. When this process was competed by 400 mM fucose, the binding was significantly slowed down, as indicated by a time constant of 978 s. The kinetics for the displacement of bound AAL by free fucose was observed with a time constant of 424 s. We conclude that this novel EIS biosensor and the applied workflow has the potential to detect FucT and other GT activities in general and further monitor protein-glycan interactions, which may be useful for the detection of pathogenic bacteria and cancer cells in future biomedical applications.

PortaDrop: A portable digital microfluidic platform providing versatile opportunities for Lab-On-A-Chip applications.

Electrowetting-on-dielectric is a decent technique to manipulate discrete volumes of liquid in form of droplets. In the last decade, electrowetting-on-dielectric systems, also called digital microfluidic systems, became more frequently used for a variety of applications because of their high flexibility and reconfigurability. Thus, one design can be adapted to different assays by only reprogramming. However, this flexibility can only be useful if the entire system is portable and easy to use. This paper presents the development of a portable, stand-alone digital microfluidic system based on a Linux-based operating system running on a Raspberry Pi, which is unique. We present "PortaDrop" exhibiting the following key features: (1) an "all-in-one box" approach, (2) a user-friendly, self-explaining graphical user interface and easy handling, (3) the ability of integrated electrochemical measurements, (4) the ease to implement additional lab equipment via Universal Serial Bus and the General Purpose Interface Bus as well as (5) a standardized experiment documentation. We propose that PortaDrop can be used to carry out experiments in different applications, where small sample volumes in the nanoliter to picoliter range need to be handled an analyzed automatically. As a first application, we present a protocol, where a droplet is consequently exchanged by droplets of another medium using passive dispensing. The exchange is monitored by electrical impedance spectroscopy. It is the first time, the media exchange caused by passive dispensing is characterized by electrochemical impedance spectroscopy. Summarizing, PortaDrop allows easy combination of fluid handling by means of electrowetting and additional sensing.

Different Frequency of Cyclic Tensile Strain Relates to Anabolic/Catabolic Conditions Consistent with Immunohistochemical Staining Intensity in Tenocytes.

Tenocytes are mechanosensitive cells intimately adapting their expression profile and hence, their phenotype to their respective mechanomilieu. The immunolocalization and expression intensity of tenogenic, anabolic and catabolic markers in tenocytes in response to in vitro mechanical loading have not been monitored by immunohistochemical staining (IHC). Thus, we investigated the association between IHC intensities, different stimulation frequencies, and tenogenic metabolism using a versatile mechanical stretcher. Primary tenocytes obtained from murine Achilles tendons were transferred to poly(dimethylsiloxane) (PDMS) elastomeric chamber. Chambers were cyclically stretched by 5% in uniaxial direction at a variation of tensile frequency (1 or 2 Hz) for 3 h. After stretching, cell physiology, IHC intensities of tendon-related markers, and protein level of the angiogenesis marker vascular endothelial growth factor (VEGF) were evaluated. Cell proliferation in tenocytes stimulated with 1 Hz stretch was significantly higher than with 2 Hz or without stretch, while 2 Hz stretch induced significantly reduced cell viability and proliferation with microscopically detectable apoptotic cell changes. The amount of scleraxis translocated into the nuclei and tenomodulin immunoreactivity of tenocytes treated with stretch were significantly higher than of non-stretched cells. The collagen type-1 expression level in tenocytes stretched at 1 Hz was significantly higher than in those cultivated with 2 Hz or without stretching, whereas the matrix metalloproteinase (MMP)-1 and MMP-13 immunoreactivities of cells stretched at 2 Hz were significantly higher than in those stimulated with 1 Hz or without stretching. The secreted VEGF-protein level of tenocytes stretched at 2 Hz was significantly higher than without stretching. Our IHC findings consistent with cell physiology suggest that appropriate stretching can reproduce in vitro short-term tenogenic anabolic/catabolic conditions and allow us to identify an anabolic stretching profile.

Microfluidic Irreversible Electroporation-A Versatile Tool to Extract Intracellular Contents of Bacteria and Yeast.

Exploring the dynamic behavior of cellular metabolism requires a standard laboratory method that guarantees rapid sampling and extraction of the cellular content. We propose a versatile sampling technique applicable to cells with different cell wall and cell membrane properties. The technique is based on irreversible electroporation with simultaneous quenching and extraction by using a microfluidic device. By application of electric pulses in the millisecond range, permanent lethal pores are formed in the cell membrane of Escherichia coli and Saccharomyces cerevisiae, facilitating the release of the cellular contents; here demonstrated by the measurement of glucose-6-phosphate and the activity of the enzyme glucose-6-phosphate dehydrogenase. The successful application of this device was demonstrated by pulsed electric field treatment in a flow-through configuration of the microfluidic chip in combination with sampling, inactivation, and extraction of the intracellular content in a few seconds. Minimum electric field strengths of 10 kV/cm for E. coli and 7.5 kV/cm for yeast S. cerevisiae were required for successful cell lysis. The results are discussed in the context of applications in industrial biotechnology, where metabolomics analyses are important.

In Situ Monitoring of Membrane Protein Insertion into Block Copolymer Vesicle Membranes and Their Spreading via Potential-Assisted Approach.

Synthosomes are polymer vesicles with transmembrane proteins incorporated into block copolymer membranes. They have been used for selective transport in or out of the vesicles as well as catalysis inside the compartments. However, both the insertion process of the membrane protein, forming nanopores, and the spreading of the vesicles on planar substrates to form solid-supported biomimetic membranes have been rarely studied yet. Herein, we address these two points and, first, shed light on the real-time monitoring of protein insertion via isothermal titration calorimetry. Second, the spreading process on different solid supports, namely, SiO2, glass, and gold, via different techniques like spin- and dip-coating as well as a completely new approach of potential-assisted spreading on gold surfaces was studied. While inhomogeneous layers occur via traditional methods, our proposed potential-assisted strategy to induce adsorption of positively charged vesicles by applying negative potential on the electrode leads to remarkable vesicle spreading and their further fusion to form more homogeneous planar copolymer films on gold. The polymer vesicles in our study are formed from amphiphilic copolymers poly(2-methyl oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl oxazoline) (PMOXA-b-PDMS-b-PMOXA). Engineered variants of the transmembrane protein ferric hydroxamate uptake protein component A (FhuA), one of the largest ß-barrel channel proteins, are used as model nanopores. The incorporation of FhuA Δ1-160 is shown to facilitate the vesicle spreading process further. Moreover, high accessibility of cysteine inside the channel was proven by linkage of a fluorescent dye inside the engineered variant FhuA ΔCVFtev and hence preserved functionality of the channels after spreading. The porosity and functionality of the spread synthosomes on the gold plates have been examined by studying the passive ion transport response in the presence of Li+ and ClO4- ions and electrochemical impedance spectroscopy analysis. Our approach to form solid-supported biomimetic membranes via the potential-assisted strategy could be important for the development of new (bio-) sensors and membranes.

The metalloproteinase ADAM15 is upregulated by shear stress and promotes survival of endothelial cells.

Reduced shear stress resulting from disturbed blood flow can impair endothelial integrity and drive the development of vascular inflammatory lesions. Metalloproteinases of the ADAM family have been implicated in the regulation of cell survival and inflammatory responses. Here we investigate the mechanism and function of ADAM15 upregulation in primary flow cultured endothelial cells. Transcriptomic analysis indicated that within the ADAM family ADAM15 mRNA is most prominently upregulated (4-fold) when endothelial cells are exposed to physiologic shear stress. This induction was confirmed in venous, arterial and microvascular endothelial cells and is associated with increased presence of ADAM15 protein in the cell lysates (5.6-fold) and on the surface (3.1-fold). The ADAM15 promoter contains several consensus sites for the transcription factor KLF2 which is also upregulated by shear stress. Induction of endothelial KLF2 by simvastatin treatment is associated with ADAM15 upregulation (1.8-fold) which is suppressed by counteracting simvastatin with geranylgeranyl pyrophosphate. KLF2 overexpression promotes ADAM15 expression (2.1-fold) under static conditions whereas KLF2 siRNA knockdown prevents ADAM15 induction by shear stress. Functionally, ADAM15 promotes survival of endothelial cells challenged by growth factor depletion or TNF stimulation as shown by ADAM15 shRNA knockdown (1.6-fold). Exposure to shear stress increases endothelial survival while additional knockdown of ADAM15 reduces survival (6.7-fold) under flow conditions. Thus, physiologic shear stress resulting from laminar flow promotes KLF2 induced ADAM15 expression which contributes to endothelial survival. The absence of ADAM15 at low shear stress or static conditions may therefore lead to increased endothelial damage and promote vascular inflammation.

Role of Substrate Surface Morphology on the Performance of Graphene Inks for Flexible Electronics.

Two-dimensional (2D) materials, such as graphene, are seen as potential candidates for fabricating electronic devices and circuits on flexible substrates. Inks or dispersions of 2D materials can be deposited on flexible substrates by large-scale coating techniques, such as inkjet printing and spray coating. One of the main issues in coating processes is nonuniform deposition of inks, which may lead to large variations of properties across the substrates. Here, we investigate the role of surface morphology on the performance of graphene ink deposited on different paper substrates with specific top coatings. Substrates with good wetting properties result in reproducible thin films and electrical properties with low sheet resistance. The correct choice of surface morphology enables high-performance films without postdeposition annealing or treatment. Scanning terahertz time-domain spectroscopy (THz-TDS) is introduced to evaluate both the uniformity and the local conductivity of graphene inks on paper. A paper-based strain gauge is demonstrated and a variable resistor acts as an on-off switch for operating an LED. Customized surfaces can thus help in unleashing the full potential of ink-based 2D materials.

Single Interdigital Transducer Approach for Gravimetrical SAW Sensor Applications in Liquid Environments.

Surface acoustic wave (SAW) devices are well known for mass-sensitive sensor applications. In biosensing applications, chemical and biochemically evoked binding processes on surfaces are detected in liquid environments using delay line or resonator sensor configurations, preferably in combination with the appropriate microfluidic devices. All configurations share the common feature of analyzing the transmission characteristic of the propagating SAW. In this paper, a novel SAW-based impedance sensor type is introduced which uses only one interdigital transducer (IDT), simultaneously as the SAW generator and the sensor element. Here, the input port reflection coefficient S11 is measured at the IDT instead of the commonly used S21 transmission forward gain parameter. Thus, a sharp and distinct peak of the S11 spectrum is obtained, enabling a comfortable direct readout of the sensor signal. Proof of the concept was gained by analyzing the specific binding of the 4-mercaptophenylacetic acid gold nanoparticles (MPA-AuNP) directly to the IDT surface. The corresponding binding kinetic of the MPA-AuNP on the functionalized gold surface has been analyzed and a sensitivity of 7.4 mΩ nM-1 has been determined.

A new microfluidic device design for a defined positioning of neurons in vitro.

A new triangle-shaped microfluidic channel system for defined cell trapping is presented. Different variants of the same basic geometry were produced to reveal the best fitting parameter combinations regarding efficiency and sensitivity. Variants with differences in the trap gap width and the inter-trap distance were analyzed in detail by Computational Fluid Dynamics simulations and in experiments with artificial beads of different sizes (30, 60, 80 µm). Simulation analysis of flow dynamics and pressure profiles revealed strongly reduced pressure conditions and balanced flow rates inside the microfluidic channels compared to commonly used systems with meandering channels. Quantitative experiments with beads showed very good trapping results in all channel types with slight variations due to geometrical differences. Highest efficiency in terms of fast trap filling and low particle loss was shown with channel types having a larger trap gap width (20 µm) and/or a larger inter-trap distance (400 µm). Here, experimental success was achieved in almost 85% to 100% of all cases. Particle loss appeared significantly more often with large beads than with small beads. A significantly reduced trapping efficiency of about 50% was determined by using narrow trap gaps and a small inter-trap distance in combination with large 80 µm beads. The combination of the same parameters with small and medium beads led to an only slight decrease in trapping efficiency (80%). All channel types were tested qualitatively with invertebrate neurons from the pond snail Lymnaea stagnalis. The systems were appropriate to trap those sensitive neurons and to keep their viability in the trapping area at the same time.

In situ Electrochemical Impedance Spectroscopy of Electrostatically Driven Selective Gold Nanoparticle Adsorption on Block Copolymer Lamellae.

Electrostatic attraction between charged nanoparticles and oppositely charged nanopatterned polymeric films enables tailored structuring of functional nanoscopic surfaces. The bottom-up fabrication of organic/inorganic composites for example bears promising potential toward cheap fabrication of catalysts, optical sensors, and the manufacture of miniaturized electric circuitry. However, only little is known about the time-dependent adsorption behavior and the electronic or ionic charge transfer in the film bulk and at interfaces during nanoparticle assembly via electrostatic interactions. In situ electrochemical impedance spectroscopy (EIS) in combination with a microfluidic system for fast and reproducible liquid delivery was thus applied to monitor the selective deposition of negatively charged gold nanoparticles on top of positively charged poly(2-vinylpyridinium) (qP2VP) domains of phase separated lamellar poly(styrene)-block-poly(2-vinylpyridinium) (PS-b-qP2VP) diblock copolymer thin films. The acquired impedance data delivered information with respect to interfacial charge alteration, ionic diffusion, and the charge dependent nanoparticle adsorption kinetics, considering this yet unexplored system. We demonstrate that the selective adsorption of negatively charged gold nanoparticles (AuNPs) on positively charged qP2VP domains of lamellar PS-b-qP2VP thin films can indeed be tracked by EIS. Moreover, we show that the nanoparticle adsorption kinetics and the nanoparticle packing density are functions of the charge density in the qP2VP domains.

Simultaneous Electrochemical Impedance Spectroscopy and Localized Surface Plasmon Resonance in a Microfluidic Chip: New Insights into the Spatial Origin of the Signal.

A novel flow-through sensor based on electrochemical impedance spectroscopy (EIS) and localized surface plasmon resonance (LSPR) for analyzing biomolecular interactions under flow and static conditions is developed and characterized. The sensor consists of a double-side gold-coated perforated polycarbonate membrane as part of a microfluidic system made of poly(dimethylsiloxane) (PDMS). LSPR and EIS measurements are carried out simultaneously by applying media changes (water to NaCl solutions), unspecific adsorption of bovine serum albumin (BSA), or specific lectin binding on glycopolymer brushes. For BSA binding at the surface, EIS sensor signals mainly contain information from the binding activities at the sensor surface at low frequencies, whereas at high frequencies the change of bulk medium is the main contribution to the EIS signal. Here, the LSPR signal corresponds with EIS signal at high frequency. In contrast, in the case of lectin binding on glycopolymer brushes (3.4 nm thick), where the binding mainly takes place in the brush layer in the vicinity of the surface, LSPR data are correlated with the EIS signals at low frequencies. This leads to the conclusion that the origin of LSPR signals strongly depends on surface coverage and can be specified by simultaneously carrying out EIS measurements.

Lectin binding studies on a glycopolymer brush flow-through biosensor by localized surface plasmon resonance.

A localized surface plasmon resonance biosensor in a flow-through configuration was applied for investigating kinetics of lectin binding to surface-grafted glycopolymer brushes. Polycarbonate filter membranes with pore sizes of 400 nm were coated with a 114-nm thick gold layer and used as substrate for surface-initiated atom-transfer radical polymerization of a glycomonomer. These grafted from glycopolymer brushes were further modified with two subsequent enzymatic reactions on the surface to yield an immobilized trisaccharide presenting brush. Specific binding of lectins including Clostridium difficile toxin A receptor domain to the glycopolymer brush surface could be investigated in a microfluidic setup with flow-through of the analytes and transmission surface plasmon resonance spectroscopy. Graphical abstract Glycopolymer brushes serve as high affinity ligands for lectin and toxin interactions in a sensitive, disposable flow-through LSPR biosensor.

Effects of Plectin Depletion on Keratin Network Dynamics and Organization.

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin ß4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and ß1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.

Hampering of the Stability of Gold Electrodes by Ferri-/Ferrocyanide Redox Couple Electrolytes during Electrochemical Impedance Spectroscopy.

In the past decades, numerous measurements have applied electrochemical impedance spectroscopy (EIS) in an electrode-electrolyte system consisting of gold electrodes and the redox couple potassium ferrocyanide/potassium ferricyanide (HCF). Yet these measurements are often hampered by false positive and negative results. Electrochemical impedance signals often display a nonlinear drift in electrolyte systems containing the HCF redox couple, which can mask the accuracy of the analysis. Thus, this Article aims to elucidate the stability and reliability of this particular electrode-electrolyte system. Here, different gold electrode cleaning treatments were compared with respect to adsorption and roughness of the surface of gold electrodes. They show substantial nonlinear long-term drifts of the charge-transfer resistance RD. In particular, the use of HCF-containing electrolytes causes adsorption and corrosion on the gold electrode surface, resulting in a nonlinear impedance behavior that depends on the incubation period as well as on electrolyte composition. Consequently, it is strongly recommended not to use HCF containing electrolytes in combination with gold electrodes.

Flexible and Stretchable Gold Microstructures on Extra Soft Poly(dimethylsiloxane) Substrates.

Stretchable gold microstructures are reliably transferred onto an extra-soft elastomeric substrate. Several major challenges, including failure-free transfer and reliable bonding with the substrate, are addressed. The simple and reproducible fabrication allows extensive study and optimization of the stretchability of meanders in terms of thickness, geometry, and substrate. The results provide new insights for designing stretchable electronics and novel routes for stretchrelated, mechanobiological cell-interface applications.

Agarose-Based Substrate Modification Technique for Chemical and Physical Guiding of Neurons In Vitro.

A new low cost and highly reproducible technique is presented that provides patterned cell culture substrates. These allow for selective positioning of cells and a chemically and mechanically directed guiding of their extensions. The patterned substrates consist of structured agarose hydrogels molded from reusable silicon micro templates. These templates consist of pins arranged equidistantly in squares, connected by bars, which mold corresponding wells and channels in the nonadhesive agarose hydrogel. Subsequent slice production with a standard vibratome, comprising the described template pattern, completes substrate production. Invertebrate neurons of locusts and pond snails are used for this application as they offer the advantage over vertebrate cells as being very large and suitable for cultivation in low cell density. Their neurons adhere to and grow only on the adhesive areas not covered by the agarose. Agarose slices of 50 µm thickness placed on glass, polystyrene, or MEA surfaces position and immobilize the neurons in the wells, and the channels guide their neurite outgrowth toward neighboring wells. In addition to the application with invertebrate neurons, the technique may also provide the potential for the application of a wide range of cell types. Long-term objective is the achievement of isolated low-density neuronal networks on MEAs or different culture substrates for various network analysis applications.