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Drug-induced cardiotoxicity arises primarily when a compound alters the electrophysiological properties of cardiomyocytes. Features of intracellular action potentials (iAPs) are powerful biomarkers that predict proarrhythmic risks. In the last decade, a number of vertical nanoelectrodes have been demonstrated to achieve parallel and minimally-invasive iAP recordings. However, the large variability in success rate and signal strength have hindered nanoelectrodes from being broadly adopted for proarrhythmia drug assessment. In this work, we develop vertically-aligned nanocrown electrodes that are mechanically robust and achieve > 99% success rates in obtaining intracellular access through electroporation. We validate the accuracy of nanocrown electrode recordings by simultaneous patch clamp recording from the same cell. Finally, we demonstrate that nanocrown electrodes enable prolonged iAP recording for continual monitoring of the same cells upon the sequential addition of four incremental drug doses. Our technology development provides an advancement towards establishing an iAP screening assay for preclinical evaluation of drug-induced arrhythmogenicity. Nanoelectrodes for measuring intracellular action potentials suffer from issues with success rate, signal strength and fabrication. Here, the authors report on a scalable technique which creates robust nanocrown electrodes with high success rates by electroporation and demonstrate the advance towards preclinical drug evaluation.

Intracellular electrophysiology is essential in neuroscience, cardiology, and pharmacology for studying cells’ electrical properties. Traditional methods like patch-clamp are precise but low-throughput and invasive. Nanoelectrode Arrays (NEAs) offer a promising alternative by enabling simultaneous intracellular and extracellular action potential (iAP and eAP) recordings with high throughput. However, accessing intracellular potentials with NEAs remains challenging. This study presents an AI-supported technique that leverages thousands of synchronous eAP and iAP pairs from stem-cell-derived cardiomyocytes on NEAs. Our analysis revealed strong correlations between specific eAP and iAP features, such as amplitude and spiking velocity, indicating that extracellular signals could be reliable indicators of intracellular activity. We developed a physics-informed deep learning model to reconstruct iAP waveforms from extracellular recordings recorded from NEAs and Microelectrode arrays (MEAs), demonstrating its potential for non-invasive, long-term, high-throughput drug cardiotoxicity assessments. This AI-based model paves the way for future electrophysiology research across various cell types and drug interactions. Intracellular recording involves puncturing the cell membrane to gain access to the cell. In this work, the authors introduce a puncture-free intracellular recording approach that leverages a deep learning model to translate extracellular recordings into intracellular signals.

Brain-on-Chip (BoC) biotechnology is emerging as a promising tool for biomedical and pharmaceutical research applied to the neurosciences. At the convergence between lab-on-chip and cell biology, BoC couples in vitro three-dimensional brain-like systems to an engineered microfluidics platform designed to provide an in vivo-like extrinsic microenvironment with the aim of replicating tissue- or organ-level physiological functions. BoC therefore offers the advantage of an in vitro reproduction of brain structures that is more faithful to the native correlate than what is obtained with conventional cell culture techniques. As brain function ultimately results in the generation of electrical signals, electrophysiology techniques are paramount for studying brain activity in health and disease. However, as BoC is still in its infancy, the availability of combined BoC–electrophysiology platforms is still limited. Here, we summarize the available biological substrates for BoC, starting with a historical perspective. We then describe the available tools enabling BoC electrophysiology studies, detailing their fabrication process and technical features, along with their advantages and limitations. We discuss the current and future applications of BoC electrophysiology, also expanding to complementary approaches. We conclude with an evaluation of the potential translational applications and prospective technology developments.

Nanoelectrode arrays (NEAs) are emerging as promising technologies for minimally‐invasive, parallel intracellular recording. These vertical electrodes, typically hundreds of nanometers in diameter and micrometers in height, provide a means for gentle electroporation and reversible membrane permeabilization to achieve intracellular recording. Prior studies have used 5–9 vertical nanostructures per recording channel to enhance device robustness and signal strength. However, this approach complicates the establishment of a one‐to‐one correspondence between cells and electrodes. In this study, devices with recording channels featuring 1‐, 3‐, 5‐, or 9‐ vertical nanocrowns electrode arrays (NcEAs) are developed in the same device. Channels with vertical nanoelectrodes of different geometries, as well as non‐vertical electrodes, such as shallow hole electrodes and large flat electrodes, are also incorporated. These measurements demonstrate that a single NcEA not only provides high‐quality iAP recordings but also excels at preserving the intracellular waveform. In contrast, non‐vertical electrodes detect intracellular‐like signals with distorted waveforms and are not suitable for cardiac intracellular recordings. These findings highlight the critical role of electrode geometry in improving the precision and reliability of intracellular recording technologies. Intracellular recording devices featuring 1‐, 3‐, 5‐, and 9‐nanocrown electrode arrays (NcEAs) per channel, along with alternative electrode geometries, are developed. Comparative analysis shows that single NcEAs provide high‐fidelity intracellular action potentials, while multi‐nanocrown and non‐vertical electrodes yield distorted signals, emphasizing the importance of nanoelectrode geometry for reliable intracellular cardiac recordings.

Realizing the full potential of organoids and assembloids to model neural development and disease will require improved methods for long-term, minimally invasive recording of electrical activity. Current technologies, such as patch clamp, penetrating microelectrodes, planar electrode arrays and substrate-attached flexible electrodes, do not allow chronic recording of organoids in suspension, which is necessary to preserve architecture. Inspired by kirigami art, we developed flexible electronics that transition from a two-dimensional to a three-dimensional basket-like configuration with either spiral or honeycomb patterns to accommodate the long-term culture of organoids in suspension. Here we show that this platform, named kirigami electronics (KiriE), integrates with and enables chronic recording of cortical organoids for up to 120 days while preserving their morphology, cytoarchitecture and cell composition. We demonstrate integration of KiriE with optogenetic and pharmacological manipulation and modeling phenotypes related to a genetic disease. Moreover, KiriE can capture corticostriatal connectivity in assembloids following optogenetic stimulation. Thus, KiriE will enable investigation of disease and activity patterns underlying nervous system assembly. Electrical activity in neural organoids is captured with a device inspired by a paper-cutting art.

Implantable sensors can be used to monitor biomechanical strain continuously. However, three key challenges need to be addressed before they can be of use in clinical practice: the structural mismatch between the sensors and tissue or organs should be eliminated; a practical suturing attachment process should be developed; and the sensors should be equipped with wireless readout. Here, we report a wireless and suturable fibre strain-sensing system created by combining a capacitive fibre strain sensor with an inductive coil for wireless readout. The sensor is composed of two stretchable conductive fibres organized in a double helical structure with an empty core, and has a sensitivity of around 12. Mathematical analysis and simulation of the sensor can effectively predict its capacitive response and can be used to modulate performance according to the intended application. To illustrate the capabilities of the system, we use it to perform strain measurements on the Achilles tendon and knee ligament in an ex vivo and in vivo porcine leg. A capacitive, fibre-like stretchable strain sensor, formed of two conductors in a double helical structure, can be combined with an inductive coil to create a wireless strain-sensing system for biomedical applications.

Proteins, nucleic acids and ions secreted from single cells are the key signalling factors that determine the interaction of cells with their environment and the neighbouring cells. It is possible to study individual ion channels by pipette clamping, but it is difficult to dynamically monitor the activity of ion channels and transporters across the cellular membrane. Here we show that a solid-state nanopore integrated in an atomic force microscope can be used for the stochastic sensing of secreted molecules and the activity of ion channels in arbitrary locations both inside and outside a cell. The translocation of biomolecules and ions through the nanopore is observed in real time in live cells. The versatile nature of this approach allows us to detect specific biomolecules under controlled mechanical confinement and to monitor the ion-channel activities of single cells. Moreover, the nanopore microscope was used to image the surface of the nuclear membrane via high-resolution scanning ion conductance measurements.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

In bottom-up neuroscience, questions on neural information processing are addressed by engineering small but reproducible biological neural networks of defined network topology in vitro. The network topology can be controlled by culturing neurons within polydimethylsiloxane (PDMS) microstructures that are combined with microelectrode arrays (MEAs) for electric access to the network. However, currently used glass MEAs are limited to 256 electrodes and pose a limitation to the spatial resolution as well as the design of more complex microstructures. The use of high density complementary metal-oxide-semiconductor (CMOS) MEAs greatly increases the spatial resolution, enabling sub-cellular readout and stimulation of neurons in defined neural networks. Unfortunately, the non-planar surface of CMOS MEAs complicates the attachment of PDMS microstructures. To overcome the problem of axons escaping the microstructures through the ridges of the CMOS MEA, we stamp-transferred a thin film of hexane-diluted PDMS onto the array such that the PDMS filled the ridges at the contact surface of the microstructures without clogging the axon guidance channels. This method resulted in 23% of structurally fully connected but sealed networks on the CMOS MEA of which about 45% showed spiking activity in all channels. Moreover, we provide an impedance-based method to visualize the exact location of the microstructures on the MEA and show that our method can confine axonal growth within the PDMS microstructures. Finally, the high spatial resolution of the CMOS MEA enabled us to show that action potentials follow the unidirectional topology of our circular multi-node microstructure.

Bottom-up neuroscience aims to engineer well-defined networks of neurons to investigate the functions of the brain. By reducing the complexity of the brain to achievable target questions, such bioassays better control experimental variables and can serve as a versatile tool for fundamental and pharmacological research. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures can improve the quality of assays. Here, we present cellulose as an astrocyte culture substrate. Astrocytes cultured on the cellulose fiber matrix thrived and formed a dense 3D network. We devised a novel co-culture platform by suspending the easy-to-handle astrocytic paper cultures above neuronal networks of low densities typically needed for bottom-up neuroscience. There was significant improvement in neuronal viability after 5 days at densities ranging from 50,000 cells/cm down to isolated cells at 1,000 cells/cm . Cultures exhibited spontaneous spiking even at the very low densities, with a significantly greater spike frequency per cell compared to control mono-cultures. Applying the co-culture platform to an engineered network of neurons on a patterned substrate resulted in significantly improved viability and almost doubled the density of live cells. Lastly, the shape of the cellulose substrate can easily be customized to a wide range of culture vessels, making the platform versatile for different applications that will further enable research in bottom-up neuroscience and drug development.