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−1 to 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.

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.

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.

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.

Humanoids are robots created with human forms or characteristics; these robots also have the potential to seamlessly interact with human beings. By replicating the appearances and functions (e.g., self-healing) of human beings, humanoids have the potential to establish more harmonic and natural human-robot interactions. Here, we propose the use of skin equivalent, a living skin model consisting of cells and extracellular matrix, as a human-like and self-healing coverage material for robots. We fabricated a three-joint robotic finger covered with skin equivalent by developing a method to cover three-dimensional objects with skin equivalent. Furthermore, inspired by the medical treatment of deeply burned skin using grafted hydrogels, we demonstrated wound repair of a dermis equivalent covering a robotic finger by culturing the wounded tissue grafted with a collagen sheet. With the above results, this research shows the potential of using skin equivalent as human-like and self-healing coverage material for robots.

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. This journal is

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.

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.

In this letter, we introduce biohybrid robots powered by skeletal muscle tissue. Culturing myoblast-laden extracellular matrix structures enables the construc-tion of skeletal muscle tissue in vitro. Biohybrid robots constructed by the integration of such fabricated muscle tissue with robot skeletons have achieved vari-ous movements, according to the configuration of the skeleton. We believe that biohybrid robots will in-creasingly become available in the field of robotics.

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.

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.

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.

Droplet Contact Method (DCM) has widely been used to form a planar lipid bilayer between a pair of aqueous droplets for measurement of the functions of membrane proteins. However, the fabrication of DCM devices was laborious, and the integration of 3D structures into the devices remains difficult. To solve these issues, we have applied stereolithography to the fabrication of DCM devices. As a model structure, a DCM device integrated with microfluidic channels was monolithically fabricated, and a nanopore-forming protein \\alpha-hemolysin was incorporated into the planar lipid bilayer while perfusing one of the droplets. We further compared several stereolithography materials to maximize the measurement performance.

This paper proposes a biohybrid pinwheel rotated by the propulsion force of microalgae, Chlamydomonas reinhardtii. To make the microalgae rotate the pinwheel larger than themselves, we designed the pinwheel to have traps holding microalgae without preventing their motions. As a result, the biohybrid pinwheel succeeded in the rotation at the average angular velocity of 0.78 rad/s.

In this paper, we present a microfluidic device that enables the efficient pairing of giant unilamellar vesicles (GUVs) and electrofusion of paired GUVs. The device consists of a hydrodynamic microarray system for the deterministic pairing of GUVs and two thick microelectrodes, made with a low melting-point fusible alloy, for applying the electric field on the paired GUVs. We achieved 75 % efficiency for pairing GUVs with the dynamic microarray device, which was designed by computing fluid resistance of the channels. We successfully performed electrofusion of the paired GUVs arrayed in the pairing channels. Furthermore, we confirmed that the lipid component and the inner solution of the paired GUVs were fused by electrofusion respectively. We believe our microfluidic device will become a useful tool for studying biological reactions in membrane compartments.

This paper reports a culture device to compress a skin-equivalent by sandwiching it between a solenoid at the top and a substrate at the bottom. The pressure to the skin-equivalent can be controlled by changing the material of the substrate. Skin-equivalents compressed on a resin substrate were likely to be penetrated while those compressed on a polydimethylsiloxane substrate were only depressed. When the height of the substrate was low, the epidermis around the compressed area was thickened, suggesting that compression induced morphological changes of the epidermis. Our device has the potential as models of pressure-related skin diseases.

We propose an organoid-trapping device to form long intestinal epithelial organoids. The device is drivable with hydrostatic pressure to gather standard organoids, and induces their fusion in the microchannel. We can control the length of the tube-like organoids by adjusting the number of standard organoids. As a demonstration, six organoids were trapped in the device and tube-like organoids were formed by their fusion within two days. Our device will provide desired length of organoids for development of a perfusable 3D intestinal organoid model.

This paper proposes an exoskeleton biohybrid robot powered by an antagonistic pair of muscles. The 3D printed exoskeleton protects the interior of the robot from the outside physically and biochemically. In a 3D printed exoskeleton, two antagonistically arranged muscles are connected to a joint of the robot. The muscles selectively contracted in the exoskeleton by applying electrical stimulations alternately, leading to rotation of the joint. We believe that this configuration will be useful for development of biohybrid robots driven in air.

This paper reports a modular tissue assembly to construct various structures, sizes, and types of tissue. Our strategy is to separately fabricate tissue modules composed of extracellular matrix and cells, then assemble them into a scaled-up tissue with the desired structure. To evaluate the fusion of the modules, we demonstrated tissue assembly through myoblast-laden modules. The fusion of the modules was simply conducted by contacting the modules to each other. We believe that our proposed method can be used for fabricating complex tissue structures with various types of cells such as application as in-vivo tissue models and cultured meat.

We propose a co-culture method of skeletal muscle tissue and tendon tissue to mimic in vivo muscle contraction with movements. Since skeletal muscle tissue was formed between tendon tissues, the muscle tissue contract with movements even under immobilization of both ends of the co-cultured tissue due to extension of the tendon tissues. As a demonstration, we confirmed that the co-cultured tissue contracted to its long-axial direction, in contrast to contraction of the skeletal muscle tissue to its short-axial direction. Therefore, we believe that the skeletal muscle-tendon tissue will be a useful model to analyze muscle movements in vitro.

In this work, we propose a method for fabricating a biohybrid actuator by combining neural cell fiber consisting of mouse neural stem cell (mNSC) [1] and muscle tissue consisting of mouse myoblasts (C2C12). Since the neural cell fiber can be fabricated long and be easily manipulated without causing damage to neurons, this method is promising for fabricating a biohybrid actuator that features complex control of muscle tissue by remote stimulation through the mNSC-laden fibers. In experiments, we confirmed that neurons extended projections from the neural cell fiber into the muscle tissue. This result suggests that a combination of neurons cultured with core-shell fibers and three-dimensional skeletal muscle tissue could be used to form a biohybrid actuator that can drive muscle tissue by stimulation of nerves.

© 2019 Monodisperse picoliter droplets have been widely used for biochemical reactors and preparation of functional microbeads for food, cosmetics, and medical industry. Although conventional microfluidic technologies enable the production of monodisperse picoliter droplets, special instruments (e.g. syringe pump) are required, leading to its poor usability and accessibility in other research fields. In this study, we propose centrifuge-based step emulsification device housed in a microtube for simple and fast generation of monodisperse picoliter droplets. Our device consists of a reservoir part for storage of the dispersed phase and a microchannel part for droplet formation. Centrifugation using a commercial centrifuge exerts excessive pressure on the dispersed phase, infusing it into a microchannel. The infused dispersed phase is pinched off, forming droplets due to the channel geometry for step emulsification. The produced droplets at the step then are transported to the bottom of the microtube due to the centrifugal force. Using this device, we achieved formation of monodisperse water-in-oil (W/O) picoliter droplets with diameters ranging from 18 to 90 μm. Moreover, we demonstrated the cell-free protein synthesis reaction in monodisperse picoliter droplets as well as the production of cell-sized glucose-responsive hydrogel microbeads. We believe that our device would be a powerful tool for simple and fast preparation of picoliter samples with high usability.

In this chapter, the recent developments of biohybrid robots composed of muscle and synthetic components are introduced. The chapter covers two major topics: (i) the preparation methods and characteristics of muscles usable as driving elements for biohybrid robots are discussed, and (ii) the actuation properties of the biohybrid robots are described. Finally, we summarize the overview and include a discussion of future directions of biohybrid robots.

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.

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.

© 2019, © 2019 Informa UK Limited, trading as Taylor & Francis Group and The Robotics Society of Japan. 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.

We proposed a fabrication method of centimeter-sized 3D tissues using a device constructed by a 3D printer. We formed channels in a cell-laden collagen gel and established inlets and outlets. We achieved perfusion of culture medium inside the gel to supply nutrients and oxygen to the cells. As a result of the perfusion, we succeeded in 7 days of culture of a centimeter-sized cell-laden construct. We believe that the method will be useful for in vitro construction of steak meat.

We propose a pneumatic bending actuator integrated with a low-melting-point alloy-based variable stiffness endoskeleton that can bend at multiple points and maintain its bent shape without power supply. Local stiffness of the soft actuator can be altered by melting or hardening the endoskeleton with electric heat applied through embedded metal wires. Bending points of the actuator can be changed by selecting different points of the endoskeleton to be melted, and the bending angle can be controlled by injected air pressure. The shape of the bent actuator is maintained by hardening the alloy even when pressure is reduced to the initial state. We demonstrate that the actuator can be bent differently with only one pneumatic actuation layer by combining multipoint bending and the shape retention function, and thus the actuator can be used for lifting, holding, and unloading an object. We believe that the simple machinery of the actuator will be useful in programming complicated motions of soft robotic fingers, fins, and tentacles.

This study describes a perfusable and stretchable culture system for a skin-equivalent. The system is comprised of a flexible culture device equipped with connections that fix vascular channels of the skin-equivalent and functions as an interface for an external pump. Furthermore, a stretching apparatus for the culture device can be fabricated using rapid prototyping technologies, which allows for easy modifications of stretching parameters. When cultured under dynamically stretching and perfusion conditions, the skin-equivalent exhibits improved morphology. The epidermal layer becomes thicker and more differentiated than that cultured without the stretching stimuli or under statically-stretched conditions, and the dermal layer was more densely populated with dermal fibroblasts than that cultured without perfusion due to the nutrient and oxygen supply by perfusion via the vascular channels. Therefore, the system is useful for the improvement and biological studies of skin-equivalents.

© 2018 Elsevier B.V. Coaxial microfluidic devices have been widely used for the preparation of monodisperse droplets and multi-layered hydrogel fibers. However, in the formation of the coaxial microfluidic devices, as the configurations of the coaxial channels become larger and more complicated, the production time is prolonged, the yield is reduced, and there are difficulties associated with rinsing the channels. In this paper, we propose three-dimensional (3D) printed microfluidic modules as connectable platforms for the preparation of coaxial microfluidic devices. Since the microfluidic modules have simple channel configurations, we can easily fabricate them with a 3D printer and rinse their channels after use. The combination of the microfluidic modules allows the formation of various types of coaxial microfluidic devices, such as axisymmetric flow-focusing devices and co-flow devices with specified dimensions for their channels. Therefore, devices comprising these microfluidic modules have succeeded in production of monodisperse droplets and hydrogel fibers with various dimensions and different numbers of layers. Furthermore, as a demonstration of biofabrication applications, we show that the device comprising the microfluidic modules enables us to fabricate a multi-layered cell-laden fiber by gelation of cell-laden collagen solutions in a multi-layered laminar flow state. We believe that the microfluidic modules will be useful for preparing coaxial microfluidic devices for various applications in analytical chemistry, biology, and tissue engineering.

This paper describes a method to construct three-dimensional (3D) contractile human skeletal muscle tissues from a cell line. The 3D tissue was fabricated as a fiber-based structure and cultured for two weeks under tension by anchoring its both ends. While myotubes from the immortalized human skeletal myocytes used in this study never contracted in the conventional two-dimensional (2D) monolayer culture, myotubes in the 3D tissue showed spontaneous contraction at a high frequency and also reacted to the electrical stimulation. Immunofluorescence revealed that the myotubes in the 3D tissues had sarcomeres and expressed ryanodine receptor (RyR) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). In addition, intracellular calcium oscillations in the myotubes in the 3D tissue were observed. These results indicated that the 3D culture enabled the myocyte cell line to reach a more highly matured state compared to 2D culture. Since contraction is the most significant feature of skeletal muscle, we believe that our 3D human muscle tissue with the contractile ability would be a useful tool for both basic biology research and drug discovery as one of the muscle-on-chips.

This study proposes a microfluidic spinning method to form alginate microfibers with branched and chained structures by controlling two streams of a sodium alginate solution extruded from a theta-glass capillary (a double-compartmented glass capillary). The two streams have three flow regimes: (i) a combined flow regime (single-threaded stream), (ii) a separated flow regime (double-threaded stream), and (iii) a chained flow regime (stream of repeating single- and double-threaded streams). The flow rate of the sodium alginate solution and the tip diameter of the theta-glass capillary are the two parameters which decide the flow regime. By controlling the two parameters, we form branched (a Y-shaped structure composed of thick parent fiber and permanently divided two thin fibers) and chained (a repeating structure of single- and double-threaded fibers with constant frequency) alginate microfibers with various dimensions. Furthermore, we demonstrate the applicability of the alginate microfibers as sacrificial templates for the formation of chain-shaped microchannels with two inlets. Such microchannels could mimic the structure of blood vessels and are applicable for the research fields of fluidics including hemodynamics.

Organs are complex systems composed of different cells, proteins and signalling molecules that are arranged in a highly ordered structure to orchestrate a myriad of functions in our body. Biofabrication strategies can be applied to engineer 3D tissue models in vitro by mimicking the structure and function of native tissue through the precise deposition and assembly of materials and cells. This approach allows the spatiotemporal control over cell-cell and cell-extracellular matrix communication and thus the recreation of tissue-like structures. In this Review, we examine biofabrication strategies for the construction of functional tissue replacements and organ models, focusing on the development of biomaterials, such as supramolecular and photosensitive materials, that can be processed using biofabrication techniques. We highlight bioprinted and bioassembled tissue models and survey biofabrication techniques for their potential to recreate complex tissue properties, such as shape, vasculature and specific functionalities. Finally, we discuss challenges, such as scalability and the foreign body response, and opportunities in the field and provide an outlook to the future of biofabrication in regenerative medicine.

This paper describes a microfluidic device for droplet electrofusion with a continuously observation. Infusing low melting point alloy into a hydrodynamic micro array device, we successfully trapped and fused a pair of droplets by applying direct current (DC) pulses. Furthermore, we confirmed that the device allowed continuous observation for sequential fusion of droplets. We believe that this droplet electrofusion device will be useful in droplet based chemical/biological reaction study.

We propose a cell-based odorant sensor using a smartphone camera. A smartphone is a desirable all-in-one device to detect fluorescence because it has a camera, a light, a battery and an image processor. To confirm applicability to odorant sensors, we added odorants to cells expressing olfactory receptors on a smartphone camera. As a result, the camera detected slight increase of fluorescence intensity. From the result, we believe that the cell-based odorant sensor using the smartphone camera will promote the development of portable odorant sensors.

We propose a coaxial multiple-layered cell-laden fiber to analyze direct cell-cell interactions under hierarchic conditions (Fig. 1). Using coaxial microfluidic devices assembled with microfluidic modules, we succeeded in preparation of hierarchic-layered cell-laden fibers with control of their dimensions and the number of layers. Because the coaxial microfluidic devices allow arrangement of different types of cells in each layer, we achieved formation of cell-cell interactions between hierarchic structures. As a demonstration, we formed layered HepG2 and 3T3 cell-laden fibers, and showed increase of albumin secretion from HepG2 cells by interaction with 3T3 cells. Therefore, we believe that the coaxial multiple-layered cell-laden fiber will be a useful tool for assay of hierarchic cell-cell interactions.

This paper reports a method for fixed-point observation of a fat fiber with mature adipocytes(Fig.1(a)). The fat fiber consisted of 3T3-L1 suspended in collagen as a core and alginate gel as a shell. We showed that the average size of lipid droplets in the microfiber was about 3 times larger than the 2D culture. By tangling the fat fiber with pillars, we succeeded in observation of the time-dependent change of the size of lipid droplets in a specific part. Owing to fixed-point observation of mature adipocytes, we believe that our method will be useful for the study of lipid metabolism.

© The Royal Society of Chemistry 2017. 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.

© 2016 Elsevier Ltd This paper describes a method for fabricating perfusable vascular channels coated with endothelial cells within a cultured skin-equivalent by fixing it to a culture device connected to an external pump and tubes. A histological analysis showed that vascular channels were constructed in the skin-equivalent, which showed a conventional dermal/epidermal morphology, and the endothelial cells formed tight junctions on the vascular channel wall. The barrier function of the skin-equivalent was also confirmed. Cell distribution analysis indicated that the vascular channels supplied nutrition to the skin-equivalent. Moreover, the feasibility of a skin-equivalent containing vascular channels as a model for studying vascular absorption was demonstrated by measuring test molecule permeation from the epidermal layer into the vascular channels. The results suggested that this skin-equivalent can be used for skin-on-a-chip applications including drug development, cosmetics testing, and studying skin biology.

© 2017 American Chemical Society. We evaluated the speed profile of self-propelled underwater oil droplets comprising a hydrophobic aldehyde derivative in terms of their diameter and the surrounding surfactant concentration using a microfluidic device. We found that the speed of the oil droplets is dependent on not only the surfactant concentration but also the droplet size in a certain range of the surfactant concentration. This tendency is interpreted in terms of combination of the oil and surfactant affording spontaneous emulsification in addition to the Marangoni effect.

© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim This paper describes a centrifuge-based device for oil-free and mass production of calcium-alginate (Ca-alginate) particles. The device is composed of four components: a tank with a glass capillary for forming sodium alginate droplets, a collecting bath with calcium chloride (CaCl 2 ) solution, a waste liquid box, and a bypass channel bridged between the collecting bath and the waste liquid box. When the device is centrifuged, extra CaCl 2 solution in the collecting bath is delivered to the waste liquid box to maintain the appropriate liquid level of CaCl 2 solution for the production of monodisperse Ca-alginate particles. The proposed device enables oil-free production of over 45 000 uniformly sized Ca-alginate particles in a single 240 s process, whereas using the conventional method with only a glass capillary, ≈1000 particles are formed within the same processing time. Because of the high biocompatibility of the oil-free process, the device is applicable to cell encapsulation in Ca-alginate particles with high cell viability, as well as the formation of a macroscopic 3D cellular structure using Ca-alginate particles covered with cells as assembly modules. These results suggest that the device can be a useful tool for preparing experimental platforms in biomedical and tissue engineering fields.

© 2016 The Royal Society of Chemistry. We propose a method for the production of a fiber-shaped three-dimensional (3D) cellular construct of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) for the quantification of the contractile force. By culturing the cardiomyocytes in a patterned hydrogel structure with fixed edges, we succeeded in fabricating hiPS-CM fibers with aligned cardiomyocytes. The fiber generated contractile force along the fiber direction due to the hiPS-CM alignment, and we were able to measure its contractile force accurately. Furthermore, to demonstrate the drug reactivity of hiPS-CM fibers, the changes in the contractile frequency and force following treatment with isoproterenol and propranolol were observed. We believe that hiPS-CM fibers will be a useful tool for pharmacokinetic analyses during drug development.

© 2016 by the authors. In this paper, we propose a balloon pump with floating valves to control the discharge flow rates of sample solutions. Because the floating valves were made from a photoreactive resin, the shapes of the floating valves could be controlled by employing different exposure patterns without any change in the pump configurations. Owing to the simple preparation process of the pump, we succeeded in changing the discharge flow rates in accordance with the number and length of the floating valves. Because our methods could be used to easily prepare balloon pumps with arbitrary discharge properties, we achieved several microfluidic operations by the integration of the balloon pumps with microfluidic devices. Therefore, we believe that the balloon pump with floating valves will be a useful driving component for portable microfluidic systems.

© 2016 The Society for Biotechnology, Japan Vessel-like channels fabricated by embedding sacrificial structures in three-dimensional (3D) cellular constructs and then removing the sacrificial structures have been proposed as a means of providing nutrition to the cells. Alginate gel fibers have been used in the design of such channels owing to their flexibility. However, these channels are closed during culture due to extensive shrinkage of the hydrogel structures when they contain certain cell types such as fibroblasts. Here, we describe a method for fabricating vessel-like channels supported by semi-permeable poly-L-lysine-alginate membrane tubes (PLL-tubes) in a collagen gel. PLL-coated alginate gel fibers were embedded in collagen gel and the inner alginate gel was removed. We were able to form channels in various designs—including branched structures—owing to the flexibility of the alginate gel fibers. Moreover, channels supported by PLL-tubes remained open without shrinkage of the collagen gel containing fibroblasts. These results demonstrate that 3D cellular constructs can be fabricated for culturing cells that would normally induce shrinkage of hydrogel structures.

Microsized cellular constructs such as cellular aggregates and cell-laden hydrogel blocks are attractive cellular building blocks to reconstruct 3D macroscopic tissues with spatially ordered cells in bottom-up tissue engineering. In this regard, microfluidic techniques are remarkable methods to form microsized cellular constructs with high production rate and control of their shapes such as point, line, and plane. The fundamental shapes of the cellular constructs allow for the fabrication of larger arbitrary-shaped tissues by assembling them. This review introduces microfluidic formation methods of microsized cellular constructs and manipulation techniques to assemble them with control of their arrangements. Additionally, we show applications of the cellular constructs to biological studies and clinical treatments and discuss future trends as their potential applications. (C) 2015 Elsevier B.V. All rights reserved.

This paper describes a liquid-filled tunable lenticular lens for switching between two-dimensional (2D) and three-dimensional (3D) images in naked-eye 3D displays. Compared with previous 2D/3D switchable displays, this tunable lenticular lens that is directly attached to a smartphone display can project both a 2D image with the original resolution of the smartphone display and a 3D image with high brightness. This lens is simply composed of transparent poly(dimethylsiloxane) (PDMS) microchannels. While the thin top membrane on the microchannels is normally flat to transmit light without deflection for displaying 2D images, applying pressure to the microchannel deforms the membrane to acquire characteristics of lenticular lenses for 3D images. We successfully demonstrate the switching between the 2D and 3D modes. We believe that our lens can be applied as a part of a portable 2D/3D naked-eye 3D display.

© 2015 Elsevier B.V. Background: Mouse pups are invaluable model animals for understanding the molecular and neural basis underlying behavioral development. Stereotaxic operations with anesthetic control are useful tools in systems neuroscience. However, there are no commercially available anesthetic or stereotaxic devices for mouse pups. Current devices have several problems such as invasive approach for stabilization, poor sanitary control, and less flexibility to combine other surgical apparatuses. New Method: Here, we developed an inhalation anesthetic device equipped with stereotaxic function for mouse pups, by using polydimethylsiloxane (PDMS). PDMS is tolerant to heat and water exposure, and soft enough to cut or make a hole. The anesthetic and the stereotaxic parts were fabricated from the three-dimensional computer-aided design (3D CAD) data obtained from the head of a real mouse pup. Results: To confirm its utility, a tracer was injected into the brain. We were able to anesthetize and stabilize pups at once in a non-invasive manner using the PDMS device. The histological staining revealed that tracer injection was successful. Our device was compatible with various types of commercial stereotaxic and anesthetic apparatuses via trimming and tube insertion, respectively. Comparison with existing method(s): To our knowledge, this is the first report of a device that can stabilize the mouse pup's head with the non-invasive manner and functions as an inhalation anesthetic device that can be sterilized. Conclusions: The present fabrication method will provide a handy and functional instrument for stereotaxic operations in animal models at various developmental stages.

A millimeter-sized neural building block (NBB) shows high versatility to form a 3D heterogeneous neural component. A millimeter-sized 3D neural network between heterogeneous neural tissues is established, and an efficient technique is then developed to observe the spatiotemporal metrological changes of single neuron in the NBB. This technique allows the visualization of axonal extension, dendritic branching, and morphological changes of presynaptic components and synapses in real time.

This paper describes a fabrication method of muscle tissue constructs driven by neurotransmitters released from activated motor neurons. The constructs consist of three-dimensional (3D) free-standing skeletal muscle fibers co-cultured with motor neurons. We differentiated mouse neural stem cells (mNSCs) cultured on the skeletal muscle fibers into neurons that extend their processes into the muscle fibers. We found that acetylcholine receptors (AChRs) were formed at the connection between the muscle fibers and the neurons. The neuron-muscle constructs consist of highly aligned, long and matured muscle fibers that facilitate wide contractions of muscle fibers in a single direction. The contractions of the neuron-muscle construct were observed after glutamic acid activation of the neurons. The contraction was stopped by treatment with curare, an neuromuscular junction (NMJ) antagonist. These results indicate that our method succeeded in the formation of NMjs in the neuron-muscle constructs. The neuron-muscle construct system can potentially be used in pharmacokinetic assays related to NMJ disease therapies and in soft-robotic actuators. (C) 2013 Elsevier Ltd. All rights reserved.

This mini-review consists of microfluidic fabrication methods of cellular spheroids and cell-laden hydrogels, and their applications for tissue engineering. Using microfluidic devices, cellular spheroids and cell-laden hydrogels with controllable design are formed reproducibly. Owing to their size uniformity, they are used as building blocks for bottom-up tissue engineering to construct uniform and arbitrarily shaped tissues. Thus, cellular spheroids and cell-laden hydrogels based on microfluidic techniques are powerful tools to create tissues for human implantation and the treatment of diseases.

A combination of photolithography and stereolithography was successfully used to fabricate a hybrid axisymmetric flow-focusing device (h-AFFD) that produces monodisperse picoliter droplets. The h-AFFD achieved the same level of hydrodynamic performance as a monolithic AFFD produced by only stereolithography from acrylic resin. Since the h-AFFD had a narrower orifice (50 or 100 mu m in diameter), created in an SU-8 sheet by photolithography, than the monolithic AFFD, we were able to produce picoliter droplets. We also succeeded in producing monodisperse droplets encapsulating a single cell without any surface modification.

A microfluidic system was used to prepare a large number of size-controlled collagen gel beads to form microtissue units, "cell beads", as tissue building blocks. By stacking cell beads into a doll-shaped silicone chamber, millimeter-thick tissue with uniform cell density was formed rapidly. The bead structure allowed the application into the 3D printing, achieving automated geometrical control of the formed tissues for the fabrication of functional complex tissues. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

This paper describes a method to produce monodisperse cell-encapsulating microgel beads composed of a self-assembling peptide gel for three-dimensional (3D) cell culture. We used a 3D microfluidic axisymmetric now-focusing device with ail external gelation method. The finely powdered salts were dispersed into a continuous phase, and the Salts induced the gelation when in contact with the peptide solution. Over 93% of the cells survived after the encapsulation, and (he cells migrated and grew Within the gels. Applications of Our cell-encapsulating beads include bead-based cell assays in drug testing and engineering tissue constructs.

This paper describes a three-dimensional microfluidic axisymmetric flow-focusing device (AFFD) fabricated using stereolithography. Using this method, we can fabricate AFFDs rapidly and automatically without cumbersome alignment needed in conventional methods. The AFFDs are able to be fabricated reproducibly with a micro-sized orifice of diameter around 250 mu m. Using this device, we are able to produce monodisperse water-in-oil (W/O) droplets with a coefficient of variation (CV) of less than 4.5%, W/O droplets with encapsulated microbes (CV < 4.9%) and oil-in-water (O/W) droplets (CV < 3.2%) without any surface modifications. The diameter of these droplets range from 54 to 244 mu m with respect to the flow rate ratio of the fluids used; these results are in good agreement with theoretical behavior. For applications of the AFFD, we demonstrate that these devices can be used to produce double emulsions and monodisperse hydrogel beads.

We present a method for forming monodisperse semi-permeable microcapsules composed of an alginate-poly-L-lysine (PLL) membrane for the observation of encapsulated cells. These microcapsules were prepared with a monolithic three-dimensional microfluidic axisymmetric flow-focusing device by an internal gelation method using glucono-1,5-lactone in order to provide mild conditions for the cells. The microcapsules were sufficiently monodisperse and robust to be trapped in a bead-based microfluidic array system for easy observation. We also confirmed that (i) the alginate-PLL membrane is semi-permeable so that cells and microorganisms cannot pass through it but nutrients and wastes can, (ii) cells are able to move freely inside the semi-permeable microcapsules, and (iii) cells can be successfully proliferated in the microcapsules.