Pillar electrodes embedded in the skeletal muscle tissue for selective stimulation of biohybrid actuators with increased contractile distance.

Electrodes are crucial for controlling the movements of biohybrid robots, but their external placement outside muscle tissue often leads to inefficient and non-selective stimulation of nearby biohybrid actuators. To address this, we propose embedding pillar electrodes within the skeletal muscle tissue, resulting in enhanced contraction of the target muscle without affecting the neighbor tissue with a 4 mm distance. We use finite element method simulations to establish a selectivity model, correlating the VIE(volume integration of electric field intensity within muscle tissue) with actual contractile distances under different amplitudes of electrical pulses. The simulated selective index closely aligns with experimental results, showing the potential of pillar electrodes for effective and selective biohybrid actuator stimulation. In experiments, we validated that the contractile distance and selectivity achieved with these pillar electrodes exceed conventional Au rod electrodes. This innovation has promising implications for building biohybrid robots with densely arranged muscle tissue, ultimately achieving more human-like movements. Additionally, our selectivity model offers valuable predictive tools for assessing electrical stimulation effects with different electrode designs.

Post-infarction Left Ventricular Free Wall Rupture Diagnosed by Contrast-Enhanced Computed Tomography: A Case Report.

A 70-year-old Japanese woman with hypertension, dyslipidemia, and diabetes mellitus complained of abdominal discomfort and vomiting and was brought to our emergency department by ambulance two days later with impaired consciousness. Her vital signs suggested shock with a heart rate of 120 bpm. Electrocardiogram and initial transthoracic echocardiography suggested an inferior wall ST-elevation myocardial infarction, but the altered consciousness was inconsistent. Contrast-enhanced computed tomography was urgently performed to further clarify the cause. It revealed pericardial effusion and apparent extravasation from the left ventricular wall, confirming the early definitive diagnosis of left ventricular free wall rupture. The patient underwent successful emergent surgical repair without sequelae. Differential diagnosis of left ventricular free wall rupture is important in patients with ST-elevation myocardial infarction and impaired consciousness. Contrast-enhanced computed tomography allows early diagnosis and treatment of this life-threatening complication.

Real-time quantitative characterization of ion channel activities for automated control of a lipid bilayer system.

This paper describes a novel signal processing method to characterize the activity of ion channels on a lipid bilayer system in a real-time and quantitative manner. Lipid bilayer systems, which enable single-channel level recordings of ion channel activities against physiological stimuli in vitro, are gaining attention in various research fields. However, the characterization of ion channel activities has heavily relied on time-consuming analyses after recording, and the inability to return the quantitative results in real time has long been a bottleneck to incorporating the system into practical products. Herein, we report a lipid bilayer system that integrates real-time characterization of ion channel activities and real-time response based on the characterization result. Unlike conventional batch processing, an ion channel signal is divided into short segments and processed during the recording. After optimizing the system to maintain the same characterization accuracy as conventional operation, we demonstrated the usability of the system with two applications. One is quantitative control of a robot based on ion channel signals. The velocity of the robot was controlled every second, which was around tens of times faster than the conventional operation, in proportion to the stimulus intensity estimated from changes in ion channel activities. The other is the automation of data collection and characterization of ion channels. By constantly monitoring and maintaining the functionality of a lipid bilayer, our system enabled continuous recording of ion channels over 2 h without human intervention, and the time of manual labor has been reduced from conventional 3 h to 1 min at a minimum. We believe the accelerated characterization and response in the lipid bilayer systems presented in this work will facilitate the transformation of lipid bilayer technology toward a practical level, finally leading to its industrialization.

Co-culture system of human skin equivalents with mouse neural spheroids.

This study describes a co-culture system of human skin equivalents (HSEs) and dorsal root ganglion (DRG) neurons. We prepared spheroids of mouse DRG neurons with or without Schwann cells (SCs). Spheroids comprising DRG neurons and SCs showed longer neurite extensions than those comprising DRG neurons alone. Neurite extension of more than 1 mm was observed from spheroids cultured inside HSEs, whereas neurite extension was primarily observed on the surface of HSEs from spheroids cultured on HSEs. We propose that our model may be a useful tool for studying neurite extension in the human skin.

Biohybrid tensegrity actuator driven by selective contractions of multiple skeletal muscle tissues.

Biohybrid robots are robots composed of both biological and artificial materials that can exhibit the unique characteristics commonly found in living organisms. Skeletal muscle tissues can be utilized as their actuators due to their flexibility and ON/OFF controllability, but previous muscle-driven robots have been limited to one-degree of freedom (DOF) or planar motions due to their design. To overcome this limitation, we propose a biohybrid actuator with a tensegrity structure that enables multiple muscle tissues to be arranged in a 3D configuration with balanced tension. By using muscle tissues as tension members of tensegrity structure, the contraction of muscle tissues can cause the movement of the actuator in multiple-DOFs. We demonstrate the fabrication of the biohybrid tensegrity actuator by attaching three cultured skeletal muscle tissue made from C2C12 cells and fibrin-based hydrogel to an actuator skeleton using a snap-fit mechanism. When we applied an electric field of more than 4 V mm-1to the skeletal muscle tissue, the fabricated actuator had a structure to tilt in multiple directions through the selective displacement of about 0.5 mm in a specific direction caused by the contractions of muscle tissue, resulting in 3D multi-DOF tilting motion. We also show that the actuator possesses superior characteristics of tensegrity structure such as stability and robustness by assessing the response of the actuator to external force. This biohybrid tensegrity actuator provides a useful platform for the development of muscle-driven biohybrid robots with complex and flexible movements.

Attosecond electron-beam technology: a review of recent progress.

Electron microscopy and diffraction with ultrashort pulsed electron beams are capable of imaging transient phenomena with the combined ultrafast temporal and atomic-scale spatial resolutions. The emerging field of optical electron beam control allowed the manipulation of relativistic and sub-relativistic electron beams at the level of optical cycles. Specifically, it enabled the generation of electron beams in the form of attosecond pulse trains and individual attosecond pulses. In this review, we describe the basics of the attosecond electron beam control and overview the recent experimental progress. High-energy electron pulses of attosecond sub-optical cycle duration open up novel opportunities for space-time-resolved imaging of ultrafast chemical and physical processes, coherent photon generation, free electron quantum optics, electron-atom scattering with shaped wave packets and laser-driven particle acceleration. Graphical Abstract.

Microfluidic Device to Manipulate 3D Human Epithelial Cell-Derived Intestinal Organoids.

In this study, we propose a microfluidic organoid-trapping device used to immobilize human intestinal organoids and apply fluidic stimuli to them. The proposed device has a microchannel with a trapping region with wall gaps between the channel walls and the bottom surface, and a constriction to clog the organoids in the channel. Since the introduced culture medium escapes from the gap, organoids can be cultured without excessive deformation by hydrostatic pressure. Owing to the characteristics of the organoid-trapping device, we succeeded in trapping human intestinal organoids in the channel. Furthermore, to demonstrate the applicability of the device for culturing intestinal organoids, we induced organoid fusion to form large organoids by aligning the organoids in the channel and applying fluidic shear stress to the organoids to regulate their surface structures. Therefore, we believe that organoid-trapping devices will be useful for investigating organoids aligned or loaded with fluidic stimulation.

3D-Printed Centrifugal Pump Driven by Magnetic Force in Applications for Microfluidics in Biological Analysis.

In recent years, microfluidic systems have been extensively utilized for biological analysis. The integration of pumps in microfluidic systems requires precise control of liquids and effort-intensive set-ups for multiplexed experiments. In this study, a 3D-printed centrifugal pump driven by magnetic force is presented for microfluidics and biological analysis. The permanent magnets implemented in the centrifugal pump synchronized the rotation of the driving and operating parts. Precise control of the flow rate and a wide range and variety of flow profiles are achieved by controlling the rotational speed of the motor in the driving part. The compact size and contactless driving part allow simple set-ups within commercially available culture dishes and tubes. It is demonstrated that the fabricated 3D-printed centrifugal pump can induce laminar flow in a microfluidic device, perfusion culture of in vitro tissues, and alignment of cells under shear stress. This device has a high potential for applications in microfluidic devices and perfusion culture of cells.

Functional analysis of human brain endothelium using a microfluidic device integrating a cell culture insert.

The blood-brain barrier (BBB) is a specialized brain endothelial barrier structure that regulates the highly selective transport of molecules under continuous blood flow. Recently, various types of BBB-on-chip models have been developed to mimic the microenvironmental cues that regulate the human BBB drug transport. However, technical difficulties in complex microfluidic systems limit their accessibility. Here, we propose a simple and easy-to-handle microfluidic device integrated with a cell culture insert to investigate the functional regulation of the human BBB endothelium in response to fluid shear stress (FSS). Using currently established immortalized human brain microvascular endothelial cells (HBMEC/ci18), we formed a BBB endothelial barrier without the substantial loss of barrier tightness under the relatively low range of FSS (0.1-1 dyn/cm2). Expression levels of key BBB transporters and receptors in the HBMEC/ci18 cells were dynamically changed in response to the FSS, and the effect of FSS reached a plateau around 1 dyn/cm2. Similar responses were observed in the primary HBMECs. Taking advantage of the detachable cell culture insert from the device, the drug efflux activity of P-glycoprotein (P-gp) was analyzed by the bidirectional permeability assay after the perfusion culture of cells. The data revealed that the FSS-stimulated BBB endothelium exhibited the 1.9-fold higher P-gp activity than that of the static culture control. Our microfluidic system coupling with the transwell model provides a functional human BBB endothelium with secured transporter activity, which is useful to investigate the bidirectional transport of drugs and its regulation by FSS.

3D printed microfluidic devices for lipid bilayer recordings.

This paper verifies the single-step and monolithic fabrication of 3D structural lipid bilayer devices using stereolithography. Lipid bilayer devices are utilized to host membrane proteins in vitro for biological assays or sensing applications. There is a growing demand to fabricate functional lipid bilayer devices with a short lead-time, and the monolithic fabrication of components by 3D printing is highly anticipated. However, the prerequisites of 3D printing materials which lead to reproducible lipid bilayer formation are still unknown. Here, we examined the feasibility of membrane protein measurement using lipid bilayer devices fabricated by stereolithography. The 3D printing materials were characterized and the surface smoothness and hydrophobicity were found to be the relevant factors for successful lipid bilayer formation. The devices were comparable to the ones fabricated by conventional procedures in terms of measurement performances like the amplitude of noise and the waiting time for lipid bilayer formation. We further demonstrated the extendibility of the technology for the functionalization of devices, such as incorporating microfluidic channels for solution exchangeability and arraying multiple chambers for robust measurement.

Skeletal muscle-adipose cocultured tissue fabricated using cell-laden microfibers and a hydrogel sheet.

The emerging interest in skeletal muscle tissue originates from its unique properties that control body movements. In particular, recent research advances in engineered skeletal muscle tissue have broadened the possibilities of applications in nonclinical models. However, due to the lack of adipose tissue, current engineered skeletal muscle tissue has the limitation of satisfying in vivo-like position and proportion of intermuscular fat. Adipose tissue within the skeletal muscle affects their functional properties. Here, a fabrication method for cocultured tissue composed of skeletal muscle and adipose tissues is proposed to reproduce the functional and morphological characteristics of muscle. By implementing prematured adipose microfibers in a myoblast-laden hydrogel sheet, both the accumulation of large lipid droplets and control of the position of adipose tissue within the skeletal muscle tissue becomes feasible. The findings of this study provide helpful information regarding engineered skeletal muscle, which has strong potential in drug screening models.

A Cylindrical Molding Method for the Biofabrication of Plane-Shaped Skeletal Muscle Tissue.

Muscle tissues can be fabricated in vitro by culturing myoblast-populated hydrogels. To counter the shrinkage of the myoblast-populated hydrogels during culture, a pair of anchors are generally utilized to fix the two ends of the hydrogel. Here, we propose an alternative method to counter the shrinkage of the hydrogel and fabricate plane-shaped skeletal muscle tissues. The method forms myoblast-populated hydrogel in a cylindrical cavity with a central pillar, which can prevent tissue shrinkage along the circumferential direction. By eliminating the usages of the anchor pairs, our proposed method can produce plane-shaped skeletal muscle tissues with uniform width and thickness. In experiments, we demonstrate the fabrication of plane-shaped (length: ca. 10 mm, width: 5~15 mm) skeletal muscle tissue with submillimeter thickness. The tissues have uniform shapes and are populated with differentiated muscle cells stained positive for myogenic differentiation markers (i.e., myosin heavy chains). In addition, we show the assembly of subcentimeter-order tissue blocks by stacking the plane-shaped skeletal muscle tissues. The proposed method can be further optimized and scaled up to produce cultured animal products such as cultured meat.

Formation of contractile 3D bovine muscle tissue for construction of millimetre-thick cultured steak.

Owing to the increase in the global demand of meat, cultured meat technology is being developed to circumvent a shortage of meat in the future. However, methods for construction of millimetre-thick bovine muscle tissues with highly aligned myotubes have not yet been established. Here, we propose a culture method for constructing 3D-cultured bovine muscle tissue containing myotubes aligned along its long-axial direction, which contracted in response to electrical stimulation. First, we optimised the composition of biomaterials used in the construction and the electrical stimulation applied to the tissue during culture. Subsequently, we fabricated millimetre-thick bovine muscle tissues containing highly aligned myotubes by accumulating bovine myoblast-laden hydrogel modules. The microbial content of the bovine muscle tissue cultured for 14 days was below the detection limit, indicating that the muscle tissues were sterile, unlike commercial meat. Therefore, the proposed construction method for bovine muscle tissues will be useful for the production of clean cultured steak meat simulating real meat.

Single-Cycle Optical Control of Beam Electrons.

We report the single-cycle optical control of a freely propagating electron beam with an isolated cycle of midinfrared light. In particular, we produce and characterize a modulated electron current with peak-cycle-specific subfemtosecond structure in time. The direct effects of the carrier-envelope phase, amplitude, and dispersion of the optical waveform on the temporal composition, pulse durations, and chirp of the free-space electron wave function demonstrate the subcycle nature of our control. These results and concept may create novel opportunities in free-electron lasers, laser-driven particle accelerators, ultrafast electron microscopy, and wherever else high-energy electrons are needed with the temporal structure of single-cycle light.

Biohybrid robot with skeletal muscle tissue covered with a collagen structure for moving in air.

Biohybrid robots composed of biological and synthetic components have been introduced to reconstruct biological functions in mechanical systems and obtain better understanding of biological designs. For example, biohybrid robots powered by skeletal muscle tissue have already succeeded in performing various movements. However, it has been difficult for the conventional biohybrid robots to actuate in air, as the skeletal muscle tissue often dries out in air and is damaged. To overcome this limitation, we propose a biohybrid robot in which the skeletal muscle tissue is encapsulated in a collagen structure to maintain the required humidity conditions when operated in air. As the skeletal muscle tissue maintains high cell viability and contractility, even after encapsulation within the collagen structure, the biohybrid robot can move in air through contractions of the skeletal muscle tissue. To demonstrate the applicability of the developed biohybrid robot, we demonstrate its use in object manipulation. In addition, to prove its capability of functionality enhancement, we show that the biohybrid robot can actuate for a long term when perfusable tubes are set inside the collagen structure; it can actuate even while culturing cells on its surface. The developed biohybrid robot composed of skeletal muscle tissue and collagen structure can be employed within platforms used to replicate various motions of land animals.

Octave-spanning single-cycle middle-infrared generation through optical parametric amplification in LiGaS2.

We report the generation of extremely broadband and inherently phase-locked mid-infrared pulses covering the 5 to 11 µm region. The concept is based on two stages of optical parametric amplification starting from a 270-fs Yb:KGW laser source. A continuum seeded, second harmonic pumped pre-amplifier in ß-BaB2O4 (BBO) produces tailored broadband near-infrared pulses that are subsequently mixed with the fundamental pump pulses in LiGaS2 (LGS) for mid-infrared generation and amplification. The pulse bandwidth and chirp is managed entirely by selected optical filters and bulk material. We find an overall quantum efficiency of 1% and a mid-infrared spectrum smoothly covering 5-11 µm with a pulse energy of 220 nJ at 50 kHz repetition rate. Electro-optic sampling with 12-fs long white-light pulses directly from self-compression in a YAG crystal reveals near-single-cycle mid-infrared pulses (32 fs) with passively stable carrier-envelope phase. Such pulses will be ideal for producing attosecond electron pulses or for advancing molecular fingerprint spectroscopy.

Editorial for the Special Issue of Selected Papers from the 9th Symposium on Micro-Nano Science and Technology on Micromachines.

The Micro-Nano Science and Technology Division of the JSME (Japan Society of Mechanical Engineers) promotes academic activities to pioneer novel research topics on microscopic mechanics [...].

Temporal Observation of Adipocyte Microfiber Using Anchoring Device.

In this paper, we propose an anchoring device with pillars to immobilize an adipocyte microfiber that has a fiber-shaped adipocyte tissue covered by an alginate gel shell. Because the device enabled the immobilization of the microfiber in a culture dish even after its transportation and the exchange of the culture medium, we can easily track the specific positions of the microfiber for a long period. Owing to the characteristics of the anchoring device, we successfully performed temporal observations of the microfiber on the device for a month to investigate the function and morphology of three-dimensional cultured adipocytes. Furthermore, to demonstrate the applicability of the anchoring device to drug testing, we evaluated the lipolysis of the microfiber's adipocytes by applying reagents with an anti-obesity effect. Therefore, we believe that the anchoring device with the microfiber will be a useful tool for temporal biochemical analyses.

Portable biohybrid odorant sensors using cell-laden collagen micropillars.

Biohybrid odorant sensors (BOSs) composed of biological materials and artificial detectors have recently attracted much attention due to their high degree of sensitivity and selectivity. Although portability is crucial for the practical use of BOSs on site, the currently used artificial detectors for biological signals are unportable. In this study, we propose a portable cell-based odorant sensor, which uses cell-laden collagen micropillars to compensate the low optical abilities of portable artificial detectors. The micropillars were composed of HEK293T cells expressing olfactory receptors, which emit a fluorescence signal based on the extent of odorant stimulation using a calcium fluorescent indicator. By stacking cells vertically in the micropillars, we achieved different levels of amplification of the fluorescence signals by varying the height of the micropillars. As a working demonstration of the portable BOS, we successfully detected different concentrations of odorants using an inexpensive web camera. The BOS was also able to distinguish the slight differences between an agonist and an antagonist. We believe that the portability of our BOS would facilitate its applications in point-of-care testing and on-site detection of hazardous materials.

Editorial for the Special Issue of Selected Papers from the 8th Symposium on Micro⁻Nano Science and Technology on Micromachines.

The Micro⁻Nano Science and Technology Division of JSME (Japan Society of Mechanical Engineers) promotes academic activities to pioneer novel research topics on microscopic mechanics. [...].