High-Aspect-Ratio Nanoelectrodes Enable Long-Term Recordings of Neuronal Signals with Subthreshold Resolution.

The further development of neurochips requires high-density and high-resolution recordings that also allow neuronal signals to be observed over a long period of time. Expanding fields of network neuroscience and neuromorphic engineering demand the multiparallel and direct estimations of synaptic weights, and the key objective is to construct a device that also records subthreshold events. Recently, 3D nanostructures with a high aspect ratio have become a particularly suitable interface between neurons and electronic devices, since the excellent mechanical coupling to the neuronal cell membrane allows very high signal-to-noise ratio recordings. In the light of an increasing demand for a stable, noninvasive and long-term recording at subthreshold resolution, a combination of vertical nanostraws with nanocavities is presented. These structures provide a spontaneous tight coupling with rat cortical neurons, resulting in high amplitude sensitivity and postsynaptic resolution capability, as directly confirmed by combined patch-clamp and microelectrode array measurements.

The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains.

Calcium (Ca2+) ions play a pivotal role in physiology and cellular signaling. The intracellular Ca2+ concentration ([Ca2+]i) is about three orders of magnitude lower than the extracellular concentration, resulting in a steep transmembrane concentration gradient. Thus, the spatial and the temporal dynamics of [Ca2+]i are ideally suited to modulate Ca2+-mediated cellular responses to external signals. A variety of highly sophisticated methods have been developed to gain insight into cellular Ca2+ dynamics. In addition to electrophysiological measurements and the application of synthetic dyes that change their fluorescent properties upon interaction with Ca2+, the introduction and the ongoing development of genetically encoded Ca2+ indicators (GECI) opened a new era to study Ca2+-driven processes in living cells and organisms. Here, we have focused on one well-established GECI, i.e., GCaMP3.0. We have systematically modified the protein with sequence motifs, allowing localization of the sensor in the nucleus, in the mitochondrial matrix, at the mitochondrial outer membrane, and at the plasma membrane. The individual variants and a cytosolic version of GCaMP3.0 were overexpressed and purified from E. coli cells to study their biophysical properties in solution. All versions were examined to monitor Ca2+ signaling in stably transfected cell lines and in primary cortical neurons transduced with recombinant Adeno-associated viruses (rAAV). In this comparative study, we provide evidence for a robust approach to reliably trace Ca2+ signals at the (sub)-cellular level with pronounced temporal resolution.

Composite Lipid Bilayers from Cell Membrane Extracts and Artificial Mixes as a Cell Culture Platform.

An artificial lipid bilayer is the closest possible model for the cell membrane. Despite that, current methods of lipid bilayer assembly and functionalization do not provide a satisfactory mimic of the cell-cell contact due to the inability to recreate an asymmetrical multicomponent system. In the current work, a method to produce an integrated solid-supported lipid bilayer combining natural extracts from cell membranes and artificially made lipid vesicles is proposed. This simple method allows delivery of transmembrane proteins and components of the extracellular matrix into the substrate. Biocompatibility of the composite natural/artificial lipid bilayers is evaluated by their interactions with the cardiomyocyte-like HL-1 cell line. Compared with fully artificial mixes, composite lipid bilayers allow cells to adhere and develop a morphologically more normal cytoskeleton.

Expressing Optogenetic Actuators Fused to N-terminal Mucin Motifs Delivers Targets to Specific Subcellular Compartments in Polarized Cells.

Optogenetics is a powerful approach in neuroscience research. However, other tissues of the body may benefit from controlled ion currents and neuroscience may benefit from more precise optogenetic expression. The present work constructs three subcellularly-targeted optogenetic actuators based on the channelrhodopsin ChR2-XXL, utilizing 5, 10, or 15 tandem repeats (TR) from mucin as N-terminal targeting motifs and evaluates expression in several polarized and non-polarized cell types. The modified channelrhodopsin maintains its electrophysiological properties, which can be used to produce continuous membrane depolarization, despite the expected size of the repeats. This work then shows that these actuators are subcellularly localized in polarized cells. In polarized epithelial cells, all three actuators localize to just the lateral membrane. The TR-tagged constructs also express subcellularly in cortical neurons, where TR5-ChR2XXL and TR10-ChR2XXL mainly target the somatodendrites. Moreover, the transfection efficiencies are shown to be dependent on cell type and tandem repeat length. Overall, this work verifies that the targeting motifs from epithelial cells can be used to localize optogenetic actuators in both epithelia and neurons, opening epithelia processes to optogenetic manipulation and providing new possibilities to target optogenetic tools.

Single-neuron mechanical perturbation evokes calcium plateaus that excite and modulate the network.

Mechanical stimulation is a promising means to non-invasively excite and modulate neuronal networks with a high spatial resolution. Despite the thorough characterization of the initiation mechanism, whether or how mechanical responses disperse into non-target areas remains to be discovered. Our in vitro study demonstrates that a single-neuron deformation evokes responses that propagate to about a third of the untouched neighbors. The responses develop via calcium influx through mechanosensitive channels and regeneratively propagate through the neuronal ensemble via gap junctions. Although independent of action potentials and synapses, mechanical responses reliably evoke membrane depolarizations capable of inducing action potentials both in the target and neighbors. Finally, we show that mechanical stimulation transiently potentiates the responding assembly for further inputs, as both gain and excitability are transiently increased exclusively in neurons that respond to a neighbor's mechanical stimulation. The findings indicate a biological component affecting the spatial resolution of mechanostimulation and point to a cross-talk in broad-network mechanical stimulations. Since giga-seal formation in patch-clamp produces a similar mechanical stimulus on the neuron, our findings inform which neuroscientific questions could be reliably tackled with patch-clamp and what recovery post-gigaseal formation is necessary.

In Vitro Simulated Neuronal Environmental Conditions Qualify Umbilical Cord Derived Highly Potent Stem Cells for Neuronal Differentiation.

The healing of neuronal injuries is still an unachieved goal. Medicine-based therapies can only extend the survival of patients, but not finally lead to a healing process. Currently, a variety of stem cell-based tissue engineering developments are the subject of many research projects to bridge this gap. As yet, neuronal differentiation of induced pluripotent stem cells (iPS), embryonic cell lines, or neuronal stem cells could be accomplished and produce functional neuronally differentiated cells. However, clinical application of cells from these sources is hampered by ethical considerations. To overcome these hurdles numerous studies investigated the potential of adult mesenchymal stem cells (MSCs) as a potential stem cell source. Adult MSCs have been approved as cellular therapeutical products due to their regenerative potential and immunomodulatory properties. Only a few of these studies could demonstrate the capacity to differentiate MSCs into active firing neuron like cells. With this study we investigated the potential of Wharton's Jelly (WJ) derived stem cells and focused on the intrinsic pluripotent stem cell pool and their potential to differentiate into active neurons. With a comprehensive neuronal differentiation protocol comprised of mechanical and biochemical inductive cues, we investigated the capacity of spontaneously forming stem cell spheroids (SCS) from cultured WJ stromal cells in regard to their neuronal differentiation potential and compared them to undifferentiated spheroids or adherent MSCs. Spontaneously formed SCSs show pluripotent and neuroectodermal lineage markers, meeting the pre-condition for neuronal differentiation and contain a higher amount of cells which can be differentiated into cells whose functional phenotypes in calcium and voltage responsive electrical activity are similar to neurons. In conclusion we show that up-concentration of stem cells from WJ with pluripotent characteristics is a tool to generate neuronal cell replacement.

Mechanical and Electronic Cell-Chip Interaction of APTES-Functionalized Neuroelectronic Interfaces.

In this work, we analyze the impact of a chip coating with a self-assembled monolayer (SAM) of (3-aminopropyl)triethoxysilane (APTES) on the electronic and mechanical properties of neuroelectronic interfaces. We show that the large signal transfer, which has been observed for these interfaces, is most likely a consequence of the strong mechanical coupling between cells and substrate. On the one hand, we demonstrate that the impedance of the interface between Pt electrodes and an electrolyte is slightly reduced by the APTES SAM. However, this reduction of approximately 13% is definitely not sufficient to explain the large signal transfer of APTES coated electrodes demonstrated previously. On the other hand, the APTES coating leads to a stronger mechanical clamping of the cells, which is visible in microscopic images of the cell development of APTES-coated substrates. This stronger mechanical interaction is most likely caused by the positively charged amino functional group of the APTES SAM. It seems to lead to a smaller cleft between substrate and cells and, thus, to reduced losses of the cell's action potential signal at the electrode. The disadvantage of this tight binding of the cells to the rigid, planar substrate seems to be the short lifetime of the cells. In our case the density of living cells starts to decrease together with the visual deformation of the cells typically at DIV 9. Solutions for this problem might be the use of soft substrates and/or the replacement of the short APTES molecules with larger molecules or molecular multilayers.

High Aspect Ratio and Light-Sensitive Micropillars Based on a Semiconducting Polymer Optically Regulate Neuronal Growth.

Many nano- and microstructured devices capable of promoting neuronal growth and network formation have been previously investigated. In certain cases, topographical cues have been successfully complemented with external bias, by employing electrically conducting scaffolds. However, the use of optical stimulation with topographical cues was rarely addressed in this context, and the development of light-addressable platforms for modulating and guiding cellular growth and proliferation remains almost completely unexplored. Here, we develop high aspect ratio micropillars based on a prototype semiconducting polymer, regioregular poly(3-hexylthiophene-2,5-diyl) (P3HT), as an optically active, three-dimensional platform for embryonic cortical neurons. P3HT micropillars provide a mechanically compliant environment and allow a close contact with neuronal cells. The combined action of nano/microtopography and visible light excitation leads to effective optical modulation of neuronal growth and orientation. Embryonic neurons cultured on polymer pillars show a clear polarization effect and, upon exposure to optical excitation, a significant increase in both neurite and axon length. The biocompatible, microstructured, and light-sensitive platform developed here opens up the opportunity to optically regulate neuronal growth in a wireless, repeatable, and spatio-temporally controlled manner without genetic modification. This approach may be extended to other cell models, thus uncovering interesting applications of photonic devices in regenerative medicine.

Light induced stimulation and delay of cardiac activity.

This article shows the combination of light activatable ion channels and microelectrode array (MEA) technology for bidirectionally interfacing cells. HL-1 cultures, a mouse derived cardiomyocyte-like cell line, transfected with channelrhodopsin were stimulated with a microscope coupled 473 nm laser and recorded with custom built 64 electrode MEAs. Channelrhodopsin induced depolarization of the cell can evoke action potentials (APs) in single cells. Spreading of the AP over the cell layer can then be measured with good spatiotemporal resolution using MEA recordings. The possibility for light induced pacemaker switching in cultures was shown. Furthermore, the suppression of APs can also be achieved with the laser. Possible applications include cell analysis, e.g. pacemaker interference or induced pacemaker switching, and medical applications such as a combined cardiac pacemaker and defibrillator triggered by light. Since current prosthesis research focuses on bidirectionality, this system may be applied to any electrogenic cell, including neurons or muscles, to advance this field.

A targeted gain-of-function screen identifies genes affecting salivary gland morphogenesis/tubulogenesis in Drosophila.

During development individual cells in tissues undergo complex cell-shape changes to drive the morphogenetic movements required to form tissues. Cell shape is determined by the cytoskeleton and cell-shape changes critically depend on a tight spatial and temporal control of cytoskeletal behavior. We have used the formation of the salivary glands in the Drosophila embryo, a process of tubulogenesis, as an assay for identifying factors that impinge on cell shape and the cytoskeleton. To this end we have performed a gain-of-function screen in the salivary glands, using a collection of fly lines carrying EP-element insertions that allow the overexpression of downstream-located genes using the UAS-Gal4 system. We used a salivary-gland-specific fork head-Gal4 line to restrict expression to the salivary glands, in combination with reporters of cell shape and the cytoskeleton. We identified a number of genes known to affect salivary gland formation, confirming the effectiveness of the screen. In addition, we found many genes not implicated previously in this process, some having known functions in other tissues. We report the initial characterization of a subset of genes, including chickadee, rhomboid1, egalitarian, bitesize, and capricious, through comparison of gain- and loss-of-function phenotypes.

Engineering Biocompatible Interfaces via Combinations of Oxide Films and Organic Self-Assembled Monolayers.

In this paper, we demonstrate that cell adhesion and neuron maturation can be guided by patterned oxide surfaces functionalized with organic molecular layers. It is shown that the difference in the surface potential of various oxides (SiO2, Ta2O5, TiO2, and Al2O3) can be increased by functionalization with a silane, (3-aminopropyl)-triethoxysilane (APTES), which is deposited from the gas phase on the oxide. Furthermore, it seems that only physisorbed layers (no chemical binding) can be achieved for some oxides (Ta2O5 and TiO2), whereas self-assembled monolayers (SAM) form on other oxides (SiO2 and Al2O3). This does not only alter the surface potential but also affects the neuronal cell growth. The already high cell density on SiO2 is increased further by the chemically bound APTES SAM, whereas the already low cell density on Ta2O5 is even further reduced by the physisorbed APTES layer. As a result, the cell density is ∼8 times greater on SiO2 compared to Ta2O5, both coated with APTES. Furthermore, neurons form the typical networks on SiO2, whereas they tend to cluster to form neurospheres on Ta2O5. Using lithographically patterned Ta2O5 layers on SiO2 substrates functionalized with APTES, the guided growth can be transferred to complex patterns. Cell cultures and molecular layers can easily be removed, and the cell experiment can be repeated after functionalization of the patterned oxide surface with APTES. Thus, the combination of APTES-functionalized patterned oxides might offer a promising way of achieving guided neuronal growth on robust and reusable substrates.

Small company mergers--good for whom?

An analysis of 105 mergers among small UK biotech companies over a 10-year period shows that the improvement of shareholder positions, rather than product pipelines or business opportunities, is the main motivation for such transactions.

MEA Recordings and Cell-Substrate Investigations with Plasmonic and Transparent, Tunable Holey Gold.

Microelectrode arrays are widely used in different fields such as neurobiology or biomedicine to read out electrical signals from cells or biomolecules. One way to improve microelectrode applications is the development of novel electrode materials with enhanced or additional functionality. In this study, we fabricated macroelectrodes and microelectrode arrays containing gold penetrated by nanohole arrays as a conductive layer. We used this holey gold to optically excite surface plasmon polaritons, which lead to a strong increase in transparency, an effect that is further enhanced by the plasmon's interaction with cell culture medium. By varying the nanohole diameter in finite-difference time domain simulations, we demonstrate that the transmission can be increased to above 70% with its peak at a wavelength depending on the holey gold's lattice constant. Further, we demonstrate that the novel transparent microelectrode arrays are as suitable for recording cellular electrical activity as standard devices. Moreover, we prove using spectral measurements and finite-difference time domain simulations that plasmonically induced transmission peaks of holey gold red-shift upon sensing medium or cells in close vicinity (<30 nm) to the substrate. Thus, we establish plasmonic and transparent holey gold as a tunable material suitable for cellular electrical recordings and biosensing applications.

Engineering of Neuron Growth and Enhancing Cell-Chip Communication via Mixed SAMs.

The interface between cells and inorganic surfaces represents one of the key elements for bioelectronics experiments and applications ranging from cell cultures and bioelectronics devices to medical implants. In the present paper, we describe a way to tailor the biocompatibility of substrates in terms of cell growth and to significantly improve cell-chip communication, and we also demonstrate the reusability of the substrates for cell experiments. All these improvements are achieved by coating the substrates or chips with a self-assembled monolayer (SAM) consisting of a mixture of organic molecules, (3-aminopropyl)-triethoxysilane and (3-glycidyloxypropyl)-trimethoxysilane. By varying the ratio of these molecules, we are able to tune the cell density and live/dead ratios of rat cortical neurons cultured directly on the mixed SAM as well as neurons cultured on protein-coated SAMs. Furthermore, the use of the SAM leads to a significant improvement in cell-chip communications. Action potential signals of up to 9.4 ± 0.6 mV (signal-to-noise ratio up to 47) are obtained for HL-1 cells on microelectrode arrays. Finally, we demonstrate that the SAMs facilitate a reusability of the samples for all cell experiments with little re-processing.

High Performance Flexible Organic Electrochemical Transistors for Monitoring Cardiac Action Potential.

Flexible and transparent electronic devices possess crucial advantages over conventional silicon based systems for bioelectronic applications since they are able to adapt to nonplanar surfaces, cause less chronic immunoreactivity, and facilitate easy optical inspection. Here, organic electrochemical transistors (OECTs) are embedded in a flexible matrix of polyimide to record cardiac action potentials. The wafer-scale fabricated devices exhibit transconductances (12 mS V-1 ) and drain-source on-to-off current ratios (≈105 ) comparable to state of the art nonflexible and superior to other reported flexible OECTs. The transfer characteristics of the devices are preserved even after experiencing extremely high bending strain and harsh crumpling. A sub-micrometer poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) layer results in a fast transport of ions between the electrolyte and the polymer channel characterized by a cut-off frequency of 1200 Hz. Excellent device performance is proved by mapping the propagation of cardiac action potentials with high signal-to-noise ratio. These results demonstrate that the electrical performance of flexible OECTs can compete with hard-material-based OECTs and thus potentially be used for in vivo applications.

How to image cell adhesion on soft polymers?

Here, we present a method to investigate cell adhesion on soft, non-conducting polymers that are implant candidate materials. Neuronal cells were grown on two elastomers (polydimethylsiloxane (PDMS) and Ecoflex®) and prepared for electron microscopy. The samples were treated with osmium tetroxide (OsO4) and uranylacetate (UrAc). Best results can be achieved when the polymers were coated with a thin iridium layer before the cell culture. This was done to emphasize the usage of soft polymers as supports for implant electrodes. A good contrast and the adhesion of the cells on soft polymers could be visualized.

Graphene Multielectrode Arrays as a Versatile Tool for Extracellular Measurements.

Graphene multielectrode arrays (GMEAs) presented in this work are used for cardio and neuronal extracellular recordings. The advantages of the graphene as a part of the multielectrode arrays are numerous: from a general flexibility and biocompatibility to the unique electronic properties of graphene. The devices used for extensive in vitro studies of a cardiac-like cell line and cortical neuronal networks show excellent ability to extracellularly detect action potentials with signal to noise ratios in the range of 45 ± 22 for HL-1 cells and 48 ± 26 for spontaneous bursting/spiking neuronal activity. Complex neuronal bursting activity patterns as well as a variety of characteristic shapes of HL-1 action potentials are recorded with the GMEAs. This paper illustrates that the potential applications of the GMEAs in biological and medical research are still numerous and diverse.

Graphene transistors for interfacing with cells: towards a deeper understanding of liquid gating and sensitivity.

This work is focused on the fabrication and analysis of graphene-based, solution-gated field effect transistor arrays (GFETs) on a large scale for bioelectronic measurements. The GFETs fabricated on different substrates, with a variety of gate geometries (width/length) of the graphene channel, reveal a linear relation between the transconductance and the width/length ratio. The area normalised electrolyte-gated transconductance is in the range of 1-2 mS·V-1·â–¡ and does not strongly depend on the substrate. Influence of the ionic strength on the transistor performance is also investigated. Double contacts are found to decrease the effective resistance and the transfer length, but do not improve the transconductance. An electrochemical annealing/cleaning effect is investigated and proposed to originate from the out-of-plane gate leakage current. The devices are used as a proof-of-concept for bioelectronic sensors, recording external potentials from both: ex vivo heart tissue and in vitro cardiomyocyte-like HL-1 cells. The recordings show distinguishable action potentials with a signal to noise ratio over 14 from ex vivo tissue and over 6 from the cardiac-like cell line in vitro. Furthermore, in vitro neuronal signals are recorded by the graphene transistors with distinguishable bursting for the first time.

Controlled Engineering of Oxide Surfaces for Bioelectronics Applications Using Organic Mixed Monolayers.

Modifying the surfaces of oxides using self-assembled monolayers offers an exciting possibility to tailor their surface properties for various applications ranging from organic electronics to bioelectronics applications. The simultaneous use of different molecules in particular can extend this approach because the surface properties can be tuned via the ratio of the chosen molecules. This requires the composition and quality of the monolayers to be controlled on an organic level, that is, on the nanoscale. In this paper, we present a method of modifying the surface and surface properties of silicon oxide by growing self-assembled monolayers comprising various compositions of two different molecules, (3-aminopropyl)-triethoxysilane and (3-glycidyloxypropyl)-trimethoxysilane, by means of in situ controlled gas-phase deposition. The properties of the resulting mixed molecular monolayers (e.g., effective thickness, hydrophobicity, and surface potential) exhibit a perfect linear dependence on the composition of the molecular layer. Finally, coating the mixed layer with poly(l-lysine) proves that the density of proteins can be controlled by the composition as well. This indicates that the method might be an ideal way to optimize inorganic surfaces for bioelectronics applications.

Implementation and application of a novel 2D magnetic twisting cytometry based on multi-pole electromagnet.

We implemented a novel 2D magnetic twisting cytometry (MTC) based on a previously reported multi-pole high permeability electromagnet, in which both the strength and direction of the twisting field can be controlled. Thanks to the high performance twisting electromagnet and the heterodyning technology, the measurement frequency has been extended to the 1 kHz range. In order to obtain high remanence of the ferromagnetic beads, a separate electromagnet with feedback control was adopted for the high magnetic field polarization. Our setup constitutes the first instrument which can be operated both in MTC mode and in magnetic tweezers (MT) mode. In this work, the mechanical properties of HL-1 cardiomyocytes were characterized in MTC mode. Both anisotropy and log-normal distribution of cell stiffness were observed, which agree with our previous results measured in MT mode. The response from these living cells at different frequencies can be fitted very well by the soft glassy rheology model.