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 (FEM) 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.

Culture insert device with perfusable microchannels enhancesin vitroskin model development and barrier function assessment.

The ever-stricter regulations on animal experiments in the field of cosmetic testing have prompted a surge in skin-related research with a special focus on recapitulation of thein vivoskin structurein vitro. In vitrohuman skin models are seen as an important tool for skin research, which in recent years attracted a lot of attention and effort, with researchers moving from the simplest 2-layered models (dermis with epidermis) to models that incorporate other vital skin structures such as hypodermis, vascular structures, and skin appendages. In this study, we designed a microfluidic device with a reverse flange-shaped anchor that allows culturing of anin vitroskin model in a conventional 6-well plate and assessing its barrier function without transferring the skin model to another device or using additional contraptions. Perfusion of the skin model through vascular-like channels improved the morphogenesis of the epidermis compared with skin models cultured under static conditions. This also allowed us to assess the percutaneous penetration of the tested caffeine permeation and vascular absorption, which is one of the key metrics for systemic drug exposure evaluation.

Lipid Bilayer Reformation Using the Wiping Blade for Improved Ion Channel Analysis.

The measurement of ion permeation activity across planar lipid bilayers is a useful technique for the functional analysis and drug evaluation of ion channels at the single-molecule level. To enhance the data throughput, parallelization of lipid bilayers is desirable. However, existing parallelized approaches face challenges in simultaneously and efficiently measuring ion channel activities under various conditions on one chip. In this study, we propose an approach to overcome these limitations by developing a device capable of repeated measurements of ion channels incorporated into individually arrayed lipid bilayers. Our device forms an array of a lipid bilayer at a micropore on a separator by merging two lipid monolayers assembled on the surface of aqueous droplets. We introduce a vertically moving, blade-shaped module─referred to as a "wiping blade"─which enables controlled disruption and reformation of the bilayer at the micropore. By optimizing the surface properties and clearance of the wiping blade, we successfully achieved repeated bilayer formation. The arrayed lipid bilayer device with the integrated wiping blade module demonstrates a 5-fold improvement in data throughput during ion channel activity measurements. Finally, we validate the practical utility of our device by evaluating the effects of an ion channel inhibitor. The developed device opens new avenues for high-throughput analysis and screening of ion channels, leading to significant advancements in drug discovery and functional studies of membrane proteins. It offers a powerful tool for researchers in the field and holds promise for accelerating drug development by targeting ion channels.

Small-artery-mimicking multi-layered 3D co-culture in a self-folding porous graphene-based film.

In vitro vessel-mimicking models have been spotlighted as a powerful tool for investigating cellular behaviours in vascular development and diseases. However, it is still challenging to create micro-scale tubular tissues while mimicking the structural features of small arteries. Here, we propose a 3D culture model of small vascular tissue using a self-folding graphene-based porous film. Vascular endothelial cells were encapsulated within the self-folding film to create a cellular construct with a controlled curvature radius ranging from 10 to 100 µm, which is comparable to the size of a human arteriole. Additionally, vascular endothelial cells and smooth muscle cells were separately co-cultured on the inner and outer surfaces of the folded film, respectively. The porous wall worked as a permeable barrier between them, affecting the cell-cell communications like the extracellular layer in the artery wall. Thus, the culture model recapitulates the structural features of a small artery and will help us better understand intercellular communications at the artery wall in physiological and pathological conditions.

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.

Mimicking Wagyu beef fat in cultured meat: Progress in edible bovine adipose tissue production with controllable fatty acid composition.

Since the current process of livestock meat production has considerable effects on the global environment, leading to high emissions of greenhouse gases, cultured meat has recently attracted attention as a suitable alternative way to acquire animal proteins. However, while most published studies on cell-cultured meat have focused on muscle tissue culture, fat production which is an important component of the process has often been neglected from this technology, even though it can enhance the meat's final taste, aroma, tenderness, texture, and palatability. In this study, we focused on bovine muscle reconstruction by monitoring and optimizing the possible expansion rate of isolated primary bovine adipose stem cells and their adipogenesis differentiation to be fully edible for cultured meat application. After approximatively 100 days of serial passages, the bovine adipose-derived stem cells, isolated from muscle tissue, underwent 57 â€‹± â€‹5 doublings in the edible cell culture medium condition. This implies that by cultivating and amplifying them, a minimum of 2.9 â€‹× â€‹1022 â€‹cells can be obtained from around 10 â€‹g of fat. It was discovered that these cells retain their adipogenesis differentiation ability for at least 12 passages. Moreover, the final lipid composition could be controlled by adjusting the fatty acid composition of the culture medium during the differentiation process, resulting in organoleptic features similar to those of real fat from muscle. This was especially so for the cis isomer oleic acid percentage, an important part of high-grade Japanese Wagyu meat. These characteristics of the primary bovine adipose-derived stem cell proliferation and adipogenesis differentiation provide valuable insights for the in vitro production of meat alternatives.

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.

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.

Millimeter-thick 3D tissues constructed by densely cellularized core-shell microfluidic bioprinting.

Recently, microfluidic bioprinting methods, which utilize microfluidic devices as printheads to deposit microfilaments, have improved printing resolution. Despite the precise placement of cells, current efforts have not succeeded in forming densely cellularized tissue within the printed constructs, which is highly desired for the biofabrication of solid-organ tissues with firm tissue consistency. In this paper, we presented a microfluidic bioprinting method to fabricate three dimension tissue constructs consisting of core-shell microfibers where extracellular matrices and cells can be encapsulated within the core of the fibers. Using the optimized printhead design and printing parameters, we demonstrated the bioprinting of core-shell microfibers into macroscale constructs and checked the viability of cells after printing. After culturing the printed tissues using the proposed dynamic culture methods, we analyzed the morphology and function of the tissues bothin vitroandin vivo. The confluent tissue morphology in the fiber cores indicates the establishment of intensive cell-cell contacts in the fiber cores, which also leads to the upregulation of the albumin-secretion function compared to the cells cultured in a 2D format. Analysis on the cell density of the confluent fiber cores indicate the formation of densely cellularized tissues with a similar level of cell density ofin-vivosolid organ tissues. In the future, better culture techniques with improved perfusion design are anticipated to enable further the fabrication of thicker tissues, which can be used as thick tissue models or implantation grafts for cell therapy.

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.

DNA-assisted selective electrofusion (DASE) of Escherichia coli and giant lipid vesicles.

Synthetic biology and cellular engineering require chemical and physical alterations, which are typically achieved by fusing target cells with each other or with payload-carrying vectors. On one hand, electrofusion can efficiently induce the merging of biological cells and/or synthetic analogues via the application of intense DC pulses, but it lacks selectivity and often leads to uncontrolled fusion. On the other hand, synthetic DNA-based constructs, inspired by natural fusogenic proteins, have been shown to induce a selective fusion between membranes, albeit with low efficiency. Here we introduce DNA-assisted selective electrofusion (DASE) which relies on membrane-anchored DNA constructs to bring together the objects one seeks to merge, and applying an electric impulse to trigger their fusion. The DASE process combines the efficiency of standard electrofusion and the selectivity of fusogenic nanostructures, as we demonstrate by inducing and characterizing the fusion of spheroplasts derived from Escherichia coli bacteria with cargo-carrying giant lipid vesicles.

Cell fiber-based 3D tissue array for drug response assay.

For the establishment of a reproducible and sensitive assay system for three-dimensional (3D) tissue-based drug screening, it is essential to develop 3D tissue arrays with uniform shapes and high cell numbers that prevent cell death in the center of the tissue. In recent years, 3D tissue arrays based on spheroids have attracted increased attention. However, they have only been used in specific tissues with hypoxic regions, such as cancer tissues, because nutrient deprivation and hypoxic regions are formed in the core as spheroids grow. Herein, we propose a method to array cell-encapsulated tube-like tissue (cell fiber (CF)) with diameters < 150 µm to prevent nutrient deprivation and hypoxia using a device that can fix the CFs, section them in uniform sizes, and transfer them to a 96-well plate. We fabricated the arrays of CF fragments from cell lines (GT1-7), cancer cells (HeLa), mouse neural stem cells (mNSCs) and differentiated mNSCs, and performed drug response assays. The array of CF fragments assessed the drug response differences among different cell types and drug responses specific to 3D tissues. The array of CF fragments may be used as a versatile drug screening system to detect drug sensitivities in various types of tissues.

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.

In vitro proliferation and long-term preservation of functional primary rat hepatocytes in cell fibers.

Primary hepatocytes are essential cellular resources for drug screening and medical transplantation. While culture systems have already succeeded in reconstituting the biomimetic microenvironment of primary hepatocytes, acquiring additional capabilities to handle them easily as well as to expand them remains unmet needs. This paper describes a culture system for primary rat hepatocytes, based on cell fiber technology, that brings scalability and handleability. Cell fibers are cell-laden core-shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium via the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated their proliferation while maintaining their viability and their hepatic specific functions for up to thirty days of subsequent culture. We then demonstrated the efficiency of proliferating primary rat hepatocytes in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Our culture system could therefore be included in innovative strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.

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.

Role of Negatively Charged Lipids Achieving Rapid Accumulation of Water-Soluble Molecules and Macromolecules into Cell-Sized Liposomes against a Concentration Gradient.

Liposomes, molecular self-assemblies resembling biological membranes, are a promising scaffold to investigate the physicochemical logic behind the complexity of living cells. Despite elaborate synthetic studies constructing cell-like chemical systems using liposomes, less attention has been paid to the proactive role of the membrane emerging as dynamics of the molecular self-assembly. This study investigated the liposomes containing anionic phospholipids by exposing them to steady flow conditions using a newly constructed automatic microfluidic observation platform. We demonstrated that the liposomes accumulated even macromolecules under the microfluidic condition without pore formation. By investigating the effect of composition of liposomes and visualizing negatively charged phospholipids upon the flow, we presumed that the external flow caused a compositional asymmetry of anionic phospholipids between the inner/outer leaflets, and the asymmetry enabled a rapid accumulation of those molecules against the concentration gradient. The current study opens new research interests regarding the nature of biological membranes under steady flow conditions.

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.

Manufacturing of animal products by the assembly of microfabricated tissues.

With the current rapidly growing global population, the animal product industry faces challenges which not only demand drastically increased amounts of animal products but also have to limit the emission of greenhouse gases and animal waste. These issues can be solved by the combination of microfabrication and tissue engineering techniques, which utilize the microtissue as a building component for larger tissue assembly to fabricate animal products. Various methods for the assembly of microtissue have been proposed such as spinning, cell layering, and 3D bioprinting to mimic the intricate morphology and function of the in vivo animal tissues. Some of the demonstrations on cultured meat and leather-like materials present promising outlooks on the emerging field of in vitro production of animal products.