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Cardiac muscle, a subtype of striated muscle composing our heart, has garnered attention as a source of autonomously driven actuators due to its inherent capability for spontaneous contraction. However, conventional cardiac biohybrid robots have utilized planar (2D) cardiac tissue consisting of a thin monolayer of cardiac myotubes with a thickness of 3–5 μm, which can generate a limited contractile force per unit footprint. In this study, 3D cardiac muscle rings were proposed as robotic actuator units. These units not only exhibit higher contractile force per unit footprint compared to their 2D counterparts due to their increased height, but they can also be integrated into desired 3D configurations. We fabricated cardiac muscle rings from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), evaluated their driving characteristics, and verified the actuation effects by integrating them with artificial components. After the 10th day from culture, the cardiac muscle rings exhibited rhythmic spontaneous contraction and increased contractile force in response to stretching stimuli. Furthermore, after constructing a centimeter-sized biohybrid self-beating actuator with an antagonistic pair structure of cardiac muscle rings, the periodic antagonistic beating motion at its tail portion was confirmed. We believe that 3D cardiac muscle rings, possessing high contractile force and capable of being positioned within limited 3D space, can be used as potent biohybrid robotic actuators. [ABSTRACT FROM AUTHOR]

The self‐assembly of amphiphilic molecules produces structures of diverse dimensions, encompassing micelles, tubules, lamellae, and vesicles. This study focuses on elucidating the controllability of amphiphilic lipid molecule self‐assembly in size and uniformity to facilitate our understanding of the molecular characteristics that correlate with the functions and attributes of the assembled structures. Electrospray deposition allows micropatterning of lipid molecules in conjunction with a conductive–nonconductive patterned substrate. The solvent in the sprayed mist undergoes evaporation during flight, leading to the deposition of dry lipids exclusively on the conductive regions of the substrate. This process enables homogeneous lipid micropatterning, effectively circumventing the coffee‐ring effect. Subsequent hydration of the lipid pattern triggers the spontaneous formation of a size‐controlled, unilamellar vesicle array on the substrate, spanning an area of a few square millimeters. The vesicles exhibits monodispersity, with a coefficient of variation below 8% for sizes ranging from 5 to 20 μm. The size‐controlled self‐assembly process is adaptable to various lipid compositions, thereby demonstrating that the molecular characteristics manifest in the morphological features appear as phase separation, budding, and curvature of vesicle membranes. The approach further validates its suitability for conducting time‐resolved analyses of molecular transport and ligand binding on the monodispersed vesicle array. [ABSTRACT FROM AUTHOR]

Planar bilayer lipid membranes (BLMs) are widely used as models for cell membranes in various applications, including drug discovery and biosensors. However, the nanometer‐thick bilayer structure, assembled through hydrophobic interactions of amphiphilic lipid molecules, makes such BLM systems mechanically and electrically unstable. In this study, we developed a device to reform BLMs using a microair bubble. The device consists of a double well divided by a separator with a microaperture, where a BLM was formed by infusing a lipid‐dispersed solvent and an aqueous droplet into each well in series. When the BLM ruptured, a microair bubble was injected from the bottom of the well to split the merged aqueous droplet at the microaperture, which resulted in the reformation of two lipid monolayers on the split droplets. By bringing the two droplets into contact, a new BLM was formed. An angled step design was introduced in the BLM device to guide the bubble and ensure the splitting of the merged droplet. We also elucidated the optimal bubble inflow rate for the reproducible BLM reformation. Using a 4‐channel parallel device, we demonstrated the individual and repeatable reformation of BLMs. Our approach will aid the development of automated and arrayed BLM systems. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Controlled Self‐AssemblyIn article number 2300543, Shoji Takeuchi and co‐workers describe the controlled self‐assembly of amphiphilic lipid molecules. Electrospray deposition allows homogeneous micropatterning of the molecules on a conductive–non‐conductive substrate, and subsequent hydration generates size‐controlled, monodisperse vesicles with various lipid mixtures. The vesicle arrays facilitate the understanding of the correlation between molecular characteristics and morphological features..By Toshihisa Osaki; Koki Kamiya; Ryuji Kawano; Kaori Kuribayashi‐Shigetomi and Shoji TakeuchiReported by Author; Author; Author; Author; Author [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Biohybrid odorant sensors that directly integrate a biological olfactory system have been increasingly studied and are suggested to be the next generation of ultrasensitive sensors by taking advantage of the sensitivity and selectivity of living organisms. In this review, we provide a detailed description of the recent developments of biohybrid odorant sensors, especially considering the requisites for their perspective of on-site applications. We introduce the methodologies to effectively capture the biological signals from olfactory systems by readout devices, and describe the essential properties regarding the gaseous detection, stability, quality control, and portability. Moreover, we address the recent progress on multiple odorant recognition using multiple sensors as well as the current screening approaches for pairs of orphan receptors and ligands necessary for the extension of the currently available range of biohybrid sensors. Finally, we discuss our perspectives for the future for the development of practical odorant sensors. [ABSTRACT FROM AUTHOR]

The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. However, its regenerative capability is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. Here, we propose a biofabrication approach to rapidly restore skeletal muscle mass, 3D histoarchitecture, and functionality. By recapitulating muscle anisotropic organization at the microscale level, we demonstrate to efficiently guide cell differentiation and myobundle formation both in vitro and in vivo. Of note, upon implantation, the biofabricated myo‐substitutes support the formation of new blood vessels and neuromuscular junctions—pivotal aspects for cell survival and muscle contractile functionalities—together with an advanced muscle mass and force recovery. Altogether, these data represent a solid base for further testing the myo‐substitutes in large animal size and a promising platform to be eventually translated into clinical scenarios. Synopsis: The regenerative capability of skeletal muscle tissue is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. By exploiting the potentials of our biofabrication approach, one can manufacture advanced cell‐laden myo‐substitutes that ultimately may restore the functionalities of severely damaged skeletal muscles in vivo. Biofabricated myo‐substitutes may represent a valid candidate for volumetric muscle loss treatment.Upon implantation, biofabricated myo‐substitutes support the formation of new blood vessels and neuromuscular junctions together with an advanced muscle mass and force recovery.The employed biofabrication approach is compatible with human primary stem cells ‐ namely pericytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this letter, we introduce biohybrid robots powered by skeletal muscle tissue. Culturing myoblast-laden extracellular matrix structures enables the construction of skeletal muscle tissue in vitro. Biohybrid robots constructed by the integration of such fabricated muscle tissue with robot skeletons have achieved various movements, according to the configuration of the skeleton. We believe that biohybrid robots will increasingly become available in the field of robotics. [ABSTRACT FROM AUTHOR]

Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules. Analysing the 3D movement of MKLP1 motors, Maruyama et al. find that dimeric C. elegans MKLP1 drives a left-handed helical motion around the microtubule with minimum protofilament switching to the right side whereas less persistent motions are driven by monomers or by heterotetramers with CYK4. These findings suggest how obstacles along crowded spindle microtubules may be avoided by CYK4 binding to MKLP1. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) is a structure located in brain capillaries that protects the brain from toxic substances in blood due to its high barrier function. The brain capillaries form a layered structure with pericytes, neurons, glial cells, and extracellular matrix proteins that is called neurovascular unit, and the structure is important to express the high barrier function of BBB. Here, we propose a method to construct a three-dimensional BBB tissue using three human BBB-composing cells, including brain endothelial cells, pericytes, and astrocytes, that mimics the in vivo BBB-like layered structure. Primary human brain endothelial cells were plated on the back side (outside) of the collagen vitrigel membrane of a culture insert, pericytes were plated on the upper side (inside), and astrocytes mixed in Matrigel were plated on the pericyte layer. The layered structure was maintained for at least 2 wk. The BBB tissue-loaded collagen vitrigel membrane can be detached from the insert frame using acetone with the tissue fixed intact and used for vertical cryosectioning to analyze the tissue interior. We also measured transendothelial electrical resistance (TEER) in the three-dimensional BBB co-culture to investigate barrier function of the brain endothelial cells. We believe that our co-culture method is useful to study engineered BBB tissues and develop reliable in vitro human BBB models in the future. [ABSTRACT FROM AUTHOR]

3D printed microfluidic devices are made of stiff and easy-to-fatigue materials and hence are difficult to have robust pneumatic valves. In this work, we describe a type of prefabricated polydimethylsiloxane (PDMS) valves, named the "Luer-lock" valve, which can be incorporated in 3D printed microfluidic devices utilizing the Luer-lock mechanism. Luer-lock design has been adopted for fluidic connections worldwide; it is facile, reliable, and inexpensive. To take advantage of the Luer-lock design, we added "valve ports" to our 3D printed microfluidic devices; prefabricated PDMS valve modules could be embedded into these valve ports, in a leak-free manner, by screwing tight the Luer-locks. In the experiment, we succeeded in fabricating pneumatic valves with a footprint diameter of 0.8 mm and verified the functionality of these valves with a shut-off pressure of 140 mbar and a maximal switching frequency of ∼1 Hz. As a demonstration, we show the serial encoding of core–shell hydrogel microfibers using the Luer-lock valves. Since the Luer-lock valves can be mass-produced and the CAD model of Luer-locks can be easily distributed, we believe that our approach has the potential to be easily adopted by researchers around the globe. [ABSTRACT FROM AUTHOR]

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB, HNF4α, ASGPR1, CYP2C19, and CYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either as in vitro models for drug screening or as implantation grafts to treat liver failure. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pseudomonas entomophila is a highly pathogenic bacterium that infects insects. It is also used as a suitable model pathogen to analyze Drosophila's innate immunity. P. entomophila's virulence is largely derived from Monalysin, a β-barrel pore-forming toxin that damages Drosophila tissues, inducing necrotic cell death. Here we report the first and efficient purification of endogenous Monalysin and its characterization. Monalysin is successfully purified as a pro-form, and trypsin treatment results in a cleaved mature form of purified Monalysin which kills Drosophila cell lines and adult flies. Electrophysiological measurement of Monalysin in a lipid membrane with an on-chip device confirms that Monalysin forms a pore, in a cleavage-dependent manner. This analysis also provides a pore-size estimate of Monalysin using current amplitude for a single pore and suggests lipid preferences for the insertion. Atomic Force Microscope (AFM) analysis displays its structure in a solution and shows that active-Monalysin is stable and composed of an 8-mer complex; this observation is consistent with mass spectrometry data. AFM analysis also shows the 8-mer structure of active-Monalysin in a lipid bilayer, and real-time imaging demonstrates the moment at which Monalysin is inserted into the lipid membrane. These results collectively suggest that endogenous Monalysin is indeed a pore-forming toxin composed of a rigid structure before pore formation in the lipid membrane. The endogenous Monalysin characterized in this study could be a desirable tool for analyzing host defense mechanisms against entomopathogenic bacteria producing damage-inducing toxins. [ABSTRACT FROM AUTHOR]

In investigations of the emergence of protocells at the origin of life, repeatable and continuous supply of molecules and ions into the closed lipid bilayer membrane (liposome) is one of the fundamental challenges. Demonstrating an abiotic process to accumulate substances into preformed liposomes against the concentration gradient can provide a clue. Here we show that, without proteins, cell-sized liposomes under hydrodynamic environment repeatedly permeate small molecules and ions, including an analogue of adenosine triphosphate, even against the concentration gradient. The mechanism underlying this accumulation of the molecules and ions is shown to involve their unique partitioning at the liposomal membrane under forced external flow in a constrained space. This abiotic mechanism to accumulate substances inside of the liposomal compartment without light could provide an energetically up-hill process for protocells as a critical step toward the contemporary cells. How small molecules could have accumulated within hypothetical protocells on the early Earth is an open question. Here automated microfluidic experiments provide evidence for abiotic accumulation of small molecules within cell-sized liposomes under hydrodynamic flow evoking a surface-mediated mechanism. [ABSTRACT FROM AUTHOR]

This paper describes a method for the rapid exchange of buffer solution during single-molecule observation. We use a simple, transparent, all-plastic one-way valve integrated on a coverslip on a flow cell. The valve is formed using a membrane made of parylene covering a microhole. It opens when a buffer solution is introduced from the microhole (the flow pushes the cover membrane) and closes when suctioning the buffer solution (the membrane is pulled back and seals the microhole to prevent the diffusion of the solution). To check valve performance, we observed the response of a rotary biomotor, F1-ATPase, for several buffer solutions in the fabricated chamber. [ABSTRACT FROM AUTHOR]

This paper proposes a nanopore-based sensor exploiting the solution exchange of a droplet-based lipid bilayer driven by a superabsorbent polymer. Biological nanopores are candidates for use in portable sensors because of their potential to recognize and detect single molecules. One of the current challenges in the development of portable nanopore sensors is the inability to achieve continuous detection. To achieve continuous detection, we have exploited the suction force of a superabsorbent polymer to drive the continuous microfluidic flow required to wash the analyte out of the droplet. The superabsorbent polymer drives the microfluidic flow without electricity, and the developed solution exchange system remains compact. To demonstrate solution exchange in the droplet containing the lipid bilayer, the concentration of heptakis(6-O-sulfo)-β-cyclodextrin was monitored in a time-dependent manner using α-hemolysin nanopores. A reduction in the concentration, attributable to solution exchange, was successfully observed. We believe that the proposed system will increase the portability and usability of nanopore sensors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This paper addresses a nanoengineering approach to create a fully three-dimensional (3D) network of living cells, providing an advanced solution to in vitro studies on either neuronal networks or artificial organs. The concept of our work relies on stackable scaffolds composed of microcontainers designed and dimensioned to favor the geometrically constrained growth of cells. The container geometry allows cells to communicate in the culture medium and freely grow their projections to form a 3D arrangement of living cells. Scaffolds are fabricated using two-photon polymerization of IP-L 780 photoresist and are coated with collagen. They are stacked by mechanical micromanipulation. Technical details of the proposed nanofabrication scheme and assembly of the modular culture environment are explained. Preliminary in vitro results using PC12 cells have shown that this structure provides a good basis for healthy cell growth for at least 16 days. Our approach is envisioned to provide tailor-made solutions of future 3D cell assemblies for potential applications in drug screening or creating artificial organs. [ABSTRACT FROM AUTHOR]

This paper describes a fabrication method and driving property of a biohybrid device with an antagonistic pair of skeletal muscle tissues and a flexible substrate. Since two skeletal muscle tissues are symmetrically arranged with the flexible substrate as the central axis, the flexible substrate deforms according to differences in their tension. In the formation of the antagonistic pair of skeletal muscle tissues, we assembled myoblast-laden hydrogel sheets and cultured them to construct a single skeletal muscle tissue on each side of the flexible substrate. We confirmed that the skeletal muscle tissue on the flexible substrate had the fundamental morphology and function of skeletal muscle. Furthermore, we made the biohybrid device actuate with deformation of the flexible substrate by selective contractions of the skeletal muscle tissues. From the deformation of the flexible substrate, we estimated the contractile force of each skeletal muscle tissue in the biohybrid device using finite element analysis. This biohybrid device, composed of an antagonistic pair of skeletal muscle tissues and a flexible substrate, can potentially be used in biological studies and pharmacokinetic assays involving the antagonistic pair of skeletal muscles. [ABSTRACT FROM AUTHOR]

We report the functional synthesis and quantification of membrane proteins―α-hemolysin from Staphylococcus aureus and the multidrug transporter EmrE from Escherichia coli―at the stabilized droplet interface bilayer using an in vitro transcription–translation system. The system developed here can expand the list of integral membrane proteins applicable for quantitative functional analysis. [ABSTRACT FROM AUTHOR]

Biohybrid robots are attracting attention as promising candidates to enhance robot applicability to studies on biological designs and in vitro construction of biological dynamic systems. Rapid progress in biohybrid robots with skeletal muscle tissues formed on a flexible substrate has enabled various types of locomotion powered by muscle tissue. However, it has been difficult to achieve high levels of both large and long-term actuations of the skeletal muscle tissues because of their spontaneous shrinkage through the course of the tissue culture. To overcome this limitation, we adapted the concept of biological systems and developed a biohybrid robot actuated by an antagonistic pair of skeletal muscle tissues. Our robot achieved large actuation (~90° of rotation of a joint) by selective contractions of the skeletal muscle tissues and a long lifetime (~1 week) by balancing tensions of the antagonistic tissues to prevent the spontaneous shrinkage. As a demonstration, we showed that our biohybrid robots allowed a pick-and-place manipulation of objects. This research may provide a platform to exceed the limitations of design in conventional biohybrid robots and replicate various lifelike movements. [ABSTRACT FROM AUTHOR]

Two-dimensional (2D) microdroplet arrays with indexed sample concentration gradients have been receiving considerable attention for high-throughput biological and medical analyses. However, the preparation of such an array by conventional methods mandates precise pipetting and/or pumping. In this paper, we introduce a method to spontaneously generate 2D-arrayed aqueous droplets using a well array, for which coarse pipetting is sufficient. The wells are connected in rows and columns via narrow channels. Aqueous solutions impregnated in the well array are split into droplets in every single well as a subsequently introduced immiscible solvent self-propagates and divides the solution at the channels. A concentration gradient of the samples can be formed across the connected solution in the well array; once droplets are generated, each droplet possesses a different sample concentration depending on its position in the array. We experimentally determined the optimal well dimensions and solvent species to obtain a high yield of droplet generation. We next demonstrated a 2D droplet array with a two-sample concentration gradient. Finally, the applicability of the system was demonstrated through a cell viability assay using a sample that induced apoptosis. We believe the proposed method contributes to simplification and miniaturization of the system to generate droplet arrays and thus is applicable to biological and medical analysis. [ABSTRACT FROM AUTHOR]

Giant liposomes or giant vesicles, with a diameter ranging from 1 to 100 μm, have been used to examine simple biophysical and biochemical processes in isolation from living cells. The gentle hydration method is a traditional method for giant liposome formation, and has been widely used because this method can easily produce giant liposomes in sufficient amounts needed for experiments. Well-defined artificial cell membranes that mimic cell membrane environments are needed to investigate complex biological functions in giant liposomes. However, in this method, it is difficult to control the liposome size, the encapsulation concentration, and the asymmetry of the phospholipid assembly. To solve these problems, many researchers have recently developed novel methods based on microfluidic technologies for giant liposome formation. In this review, we provide an overview of giant liposome formation using microfluidic technologies. We also summarize the properties of each microfluidic technology in giant liposome formation, including its effects on the monodispersion, encapsulation efficiency, size range, and asymmetry of membranes. Moreover, we introduced biological applications of the giant liposomes formed by the microfluidic technologies. [ABSTRACT FROM AUTHOR]

A pesticide vapor sensor was developed using an agarose gel-based chip containing a nanopore sensing system. Vaporized omethoate was detected by the absorption into the gel, the complex formation with a DNA aptamer, and its obstruction at the nanopore. This strategy is applicable to other vapors, expanding the versatility of nanopore sensors. [ABSTRACT FROM AUTHOR]

DNA logic circuits utilizing DNA hybridization and/or enzymatic reactions have drawn increasing attention for their potential applications in the diagnosis and treatment of cellular diseases. The compartmentalization of such a system into a microdroplet considerably helps to precisely regulate local interactions and reactions between molecules. In this study, we introduced a relay approach for enabling the transfer of DNA from one droplet to another to implement multi-step sequential logic operations. We proposed electrical fusion and mechanical splitting of droplets to facilitate the DNA flow at the inputs, logic operation, output, and serial connection between two logic gates. We developed Negative-OR operations integrated by a serial connection of the OR gate and NOT gate incorporated in a series of droplets. The four types of input defined by the presence/absence of DNA in the input droplet pair were correctly reflected in the readout at the Negative-OR gate. The proposed approach potentially allows for serial and parallel logic operations that could be used for complex diagnostic applications. [ABSTRACT FROM AUTHOR]