We have developed a microfluidic channel that can be combined with commercially available culture inserts with collagen vitrigel membranes, and have succeeded in reproducing the mechanical stimulation associated with blood flow in vivo by applying a flow of culture medium to cells cultured on vitrigel membranes. The morphology of co-cultured tissues consisting of myofibers and vascular endothelial cells was confirmed to be changed by the flow of culture medium. Furthermore, we found that the flow stimulation changed functions of co-cultured tissues consisting of hepatocytes and vascular endothelial cells, such as albumin production and albumin transfer to the flow, indicating that the proposed microfluidic system is useful as a model of vascular structure.

We succeeded in fabricating a 3D tissue array suitable for drug screening to analyze the effects of a vast array of drugs on neural tissue in a short time. The 3D tissue arrays constructed using nerve tissue with a tubular-like structure had the following characteristics: no cell death was observed at the tissue center, the number of cells was uniform, there were enough cells for the assay, and the shape was uniform. The drug assay using this 3D tissue array was able to pass the acceptance criteria for drug screening. It was also suggested that the assay could be used to evaluate cell proliferation as well as cell death. We also established a perfusion culture system for neural tissue and vascular endothelial cells. In the future, this system will be used to search for novel drugs for neural diseases.

Cell fiber manufacturing methods has been further explored. Shape control (straight, spiral and chain fibers) and 3D-printing of the cell fibers were achieved. We also created a database of the construction and culture conditions for various types of cell fibers. As an application, muscle cell fibers were shown to be used as a bio-actuator, which suggested that cell fiber technology could contribute to bio-robotics. In addition, we showed that lotus root-shaped cell fibers as a graft could be taken out without adhesion even after one year. These achievements have built a new foundation for cell fiber technology.

In this study, we showed that motor neurons and skeletal muscle fibers were connected by culturing the motor neurons on a human skeletal muscle tissue, and the muscle tissue was contractible according to transmitted signals from the motor neurons. In addition, we succeeded in formation of a skeletal muscle tissue actuator capable of muscle contractions with large contraction amount by arranging a pair of skeletal muscle tissues via a joint. This actuator with skeletal muscle tissues can control rotation of the joint according to contractions of each skeletal muscle tissue, and can manipulate objects by moving an arm attached to the joint with the muscle contraction.

Using microfluidic device mimicking human placental barrier structure, we have previously reported that fluid shear stress (FSS) induced microvilli formation via the activation of transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel in human placental trophoblastic cells. Here we found that certain type of mesenchymal cells also exhibited the similar mechanobiological responses observed in the placental trophoblastic cells. Furthermore, we established the mutant TRPV6 mouse lines harboring stop codon mutation in the coding sequence. Histological or ultrastructural analyses of TRPV6-expressing tissues will provide us with useful information to know the mechanobiological roles of TRPV6 in tissue formation and maintenance.

In this research, we constructed three-dimensional (3D) tissue shape control of neural stem cells (NSCs) tissues by using closed agarose microchambers for in vitro effective differentiation induction of neurons. As a device for controlling the 3D tissue shape, we first prepared the agarose hydrogel microchambers and sheet. By closing the agarose microchambers with an agarose sheet, lane-shaped NSCs tissues were successfully induced differentiation to neurons and glial cells while maintaining their 3D tissue shape. The thin neural tissues in which neurons were uniformly distributed at high density, was successfully fabricated. We also fabricated the neural microfiber from the human iPS-derived neural stem cells and co-cultured with HUVEC. We successfully observed the vascularization of HUVEC in the 3D fibrin gel with human neural tissues. Finally, we observed that neuronal degeneration in 3D neural tissues by the exposure of glutamic acid.

Skeletal muscle have gathered attentions as a linear actuator because its contractions are sychronized with electrical stimulations, and the power of the contraction depends on the amount of electricity. Due to high energy-conversion efficiency of skeletal muscles, many researchers have proposed bioactuators using in vitro constructed skeletal muscle tissues; conventional bioactuators achieved to walk on a Petri dish. However, the conventional bioactuators were driven only in culture medium and cannot work in air. In contrast, in vivo actuators with skeletal muscles such as arms and legs can be driven in air. Here, to mimic the in vivo properties, we proposed skeletal muscle actuators covered with cellular tissues. As a result, we succeeded in construction of curved skin tissues with vessel structures by fixation of their edges with anchors. Furthermore, we achieved driving skeletal muscle actuators in air by covering them with curved cellular tissues.

We have developed cell-cultured microfabricated microplates for manipulating adherent cells. We succeeded in three-dimensional manipulation of the adherent cells by handling the microplates with micro-tweezers, fluid flows, magnetic fields and cell traction force. In addition, we have also developed cell-cultured microplates connected with hinges, and constructed three-dimensional cellular structures by folding the microplates by the cell traction force. By embedding permalloy pieces in the cell-cultured microplates, we achieved magnetic handling of the cells on the microplates without physical contacts to both cells and microplates. Using the magnetized microplates, we developed a vertical confocal observation system. This system enables us to observe the boundary of cell membrane at a high-resolution. These progresses will contribute to construction of three-dimensional cellular structures, analysis of cellular interaction, and high-resolution observation of cells.

本研究における平成26年度の目的は、継続的に収縮運動可能な骨格筋アクチュエータを確立することである。生体外において構築された骨格筋は、臓器や動物実験の代替品としての移植医療や創薬への応用だけでなく、人工食肉や生体模倣ロボットの駆動素子として工学分野への応用が期待されている。これまでの研究で、体外での骨格筋の構築方法は確立されてきた。しかし、従来方法で作製された骨格筋アクチュエータは培養に伴って短縮や変形が生じ、継続的な収縮運動が困難であった。
そこで本年度では、腱様構造を有する拮抗筋付き骨格筋アクチュエータを構築して継続的な骨格筋の収縮運動を実現を目指した。これまでの研究結果より、大きな収縮運動を継続的に達成するためには、 (a)駆動部が正確なリンク機構であること、(b)骨格筋が変形せずに直線的に収縮運動が可能であること、(c)拮抗筋構造を実現できること、の3点を満たす必要がある。
本年度の研究成果として、(a)骨格筋を培養可能なリンク機構を3Dプリンタにより作製できることを示した。3Dプリンタで作製したリンク機構を有するデバイス上に1μm以上の厚みのポリマー膜と糖たんぱく質をコートすることで、デバイス上での骨格筋線維束の培養を実現した。(b)骨格筋の直線的な収縮運動を実現するために、腱様構造が効果的なことを明らかにした。骨格筋の片端部とリンク機構部を繋ぐ部分に厚さ2μmのポリマー膜を設けることで、リンク機構の回転運動に伴う回転方向の変形を吸収させ、骨格筋線維束の直線的な収縮運動に成功した。(c)拮抗筋構造の構築には、申請者提案の筋芽細胞含有ハイドロゲルシートをデバイスの表・裏の両面から積層することで実現した。
本デバイスにおいて拮抗筋が収縮運動することにより、約90度の大きなリンク機構の回転運動が実現できており、バイオアクチュエータや薬物動態試験などへの応用が期待できる。

© 17CBMS-0001. This paper reports a fabrication method for heterogeneous 3D structures by assembly of alginate gel modules (Fig. 1(a)). The alginate gel modules are connected by adding 'cationic nanoparticle' (CNP) solution because the CNP interacts with the anionic surface of alginate gels. We show that CNP has high biocompability to human malignant epithelial cells (HeLa). Using CNPs as adhesive, we connected alginate gel plates and confirmed the connection by stretching the plate. As a demonstration, we achieved to make a structure containing HeLa cells. We believe that our method is suitable for fabrication of 3D cell-laden structures in tissue engineering.

© 17CBMS-0001. We report formation of a three-dimensional tissue-like structure achieved by adhesion of massive liposomes. Liposomes generated by the gentle hydration method were assembled by centrifugation in a tapered mold, leading to compact aggregation of the liposomes. The liposomes then adhered to each other by avidin-biotin binding to form a liposome-based tissue-like structure. We successfully observed that the massive liposomes, which kept an original liposomal shape, were closely packed and adhered into the doll-shaped mold. We believe the proposed method to form the liposome-based tissue-like structure is applicable to various fields including tissue engineering, since liposomes can be utilized for diverse applications from simple biochemical reactor to protocell model.

© 2020 IEEE. This paper proposes a device composed of nozzle array and meshed anchors for construction of a three-dimensional (3D) muscle tissue. The nozzle array and the meshed anchors provide perfusability and ability to fix the tissue to the device, respectively. For investigation of the perfusability, we formed perfusable channels (300 μm in diameter) in a C2C12 muscle tissue and confirmed that the perfusion prevented its central necrosis. To check the fixation ability, we confirmed that meshed anchors prevented the muscle tissue from detaching the nozzles caused by intrinsic shrinkage of the tissue. As a result, we achieved construction of muscle tissue in 2.8 mm thickness with cellular orientation.

© 2020 IEEE. This paper reports a device for applying cyclic stretch and fluidic shear stress to cells toward construction of a cell barrier model. The device is composed of a culture insert with a thin porous membrane and a microfluidic channel. By fixing the culture insert to the device mechanically, there were no liquid leakages when applying both cyclic stretch and fluidic shear stress. Moreover, the culture insert was detachable from the device, leading to the easy observation of cultured cells. As a results of device characterization, we confirmed that 10% cyclic stretch and about 20 dyne/cm2 fluidic shear stress could be applied to the membrane. Therefore, we believe that our device will be a useful platform for evaluating cell response to mechanical stimulus as a cell barrier model.

© 17CBMS-0001. We propose a heterogeneous cell-laden photo-crosslinkable hydrogel array toward the detection of multiple odorant substances. We fabricated alternative patterns of cell-laden pillars containing two different cells by crosslinking hydrogel. To verify applicability to odorant sensor, we encapsulated cells expressing olfactory receptor in pillars. As a result, cells in pillars were alive and emitted fluorescence from calcium indicator for odorants. From these results, we believe that our heterogeneous cell-laden hydrogel array will promote the development of cell based odorant sensors.

© 17CBMS-0001. We propose a three-dimensional (3D) culture for anchorage-dependent cell expansion by using thickness-controlled microcarrier aggregates. Myoblast cell line, C2C12 cells and microcarriers (MCs) were encapsulated in fiber-shaped collagen-alginate gel coated by poly-L-lysine (PLL). Addition of alginate lyase allowed for the adhesion of encapsulated cells on immobilized MCs. The cells were expanded with high viability in the fiber-shaped collagen gel encapsulating MCs (termed MC fiber). Thus, we believe that the culture system can be applicable for large-scale expansion of mammalian cells.

© 17CBMS-0001. This paper reports a plain method for constructing in vitro 3D microvessels by sandwich of two cell-plated collagen gel disks. The disks were grooved in advance for cells to be plated on and to construct a tube structure after two disks were overlaid with the cell-plated sides facing each other. In this study, we demonstrated that this simple strategy worked to make a lumen structure covered with human umbilical vein endothelial cells (HUVECs) inside the collagen gel with high success rates and in shorter days than other existing methods.

© 17CBMS-0001. We propose connectable microfluidic modules as platforms for coaxial microfludics to form monodisperse droplets and layered hydrogel fibers without specific microfluidic devices according to the intended use. (Fig. 1). By integration of the connectable modules, we succeeded in preparation of various typed coaxial microfluidic devices: axisymmetric flow-focusing device (AFFD) and multiple coaxial laminar-flow device (CLFD). Using the integrated AFFDs and the integrated CLFD, we achieved formation of monodisperse water-in-oil (W/O) droplets and multiple layered hydrogel fibers including cell-laden microfibers, respectively. The results indicate that the connectable microfluidic modules are available to prepare coaxial microfluidic devices for cell manipulation and tissue engineering.

© 17CBMS-0001. This paper evaluated dynamics of flows for formation of complex gel fibers through θ glass tubes. We discovered that when sodium alginate solution was extruded into calcium chloride solution through a θ glass tube, flow modes of the solution (combined mode, separated mode and chained mode) varied in 7 steps in accordance with parameters: concentration of calcium chloride solution, tip diameters of θ glass tubes and flow rates of sodium alginate solution. By controlling these parameters, we succeeded in formation of complex alginate gel fibers: branched fibers and chained fibers. We believe that this technique will contribute to fabrication of complex vessel-like channels.

© 17CBMS-0001. This paper describes a method to fabricate a perfusable collagen-based sponge-like scaffold (collagen sponge). We lyophilized collagen solution in a culture device, and removed a wire embedded in the resultant collagen sponge to make a vascular channel. By connecting the device with an external pump, the sponge was able to be infused with medium. Moreover, we achieved formation of capillary-like structures coated with dermal fibroblasts (NHDF) and endothelial cells (HUVEC) by inoculating the cells via the channel and culturing the sponge under perfused condition. According to these results, we believe that our sponge will be a platform for perfusable vascularized 3D cellular constructs contributing to drug test and regenerative medicine.

© 2019 IEEE. We propose a method for in situ continuous and dynamic glucose monitoring in cultured skeletal muscle tissue using a glucose responsive hydrogel fiber. In the skeletal muscle tissue, the fiber emits fluorescence according to glucose concentration. In addition, the skeletal muscle tissue showed contractility even in cultured with the fiber. Therefore, from measurement of the fluorescence intensity when inducing muscle contractions by applying electrical pulses, we achieved detection of increase of glucose absorption in the skeletal muscle tissue caused by its contractions. Thus, we believe that the skeletal muscle tissue containing the glucose responsive hydrogel fiber will be a useful tool for evaluation of muscle glucose absorption without tissue lysis, in contrast to conventional method needing tissue lysis.

© 2019 IEEE. We propose a centrifuge-based microfluidic device housed in a standard microtube for generation of monodisperse microdroplets from tens of \mu \mathrm{l} sample volume. Centrifugation drives overpressure of a dispersed phase, thus infuses the dispersed phase into a microchannel. Because of the channel geometry for step emulsification, the infused dispersed phase is pinched off forming droplets. Then, the droplets accumulate on the bottom of the microtube by centrifugal force. We succeeded in generation of monodisperse droplets with a diameter of 130\ \mu\mathrm{m}(\mathrm{CV} < 2\%) by operation of centrifuge. Since the device can produce droplets in a microtube without complex experimental setup (e.g. syringe pump), it will be a powerful tool for simple generation of droplets for droplet-based applications.

We propose pneumatically driven PDMS micropillars (PDMS actuator) for the investigation of intercellular communication at a single-cell level. Cells usually organize large and complex networks. For detailed investigation of intercellular communication, downsizing the network is important. In this device, cell-sized micropillars are actuated by air pressure so that the adjacent cells on micropillars contact each other at an arbitrary timing. This actuator is also able to give mechanical stretch to coupled cells. We believe that this technology would contribute to the study of single-cell analysis of intercellular communication and the relationship between intercellular communication and mechanical stress.

This conference proceeding reports a system for measuring transendothelial electrical resistance (TEER) of 3D tubular vascular channel as an index of endothelial barrier integrity. We introduced an electrodes-pair into the channel inside a collagen sgel in a culture device, and set a counter electrodes-pair parallel to the channel (Fig. 1). By connecting the electrodes with a volt-ohm meter, TEER measurement of the vascular channel coated with endothelial cells (HUVECs) was achieved. Moreover, by perfusing the channel with culture medium via the connection of the device, the influence of the perfusion on the TEER was measured during cultivation. According to these results, we believe that our system will be a useful platform for drug permeability assay utilizing the 3D channel.

We propose a gaseous odorant detection system by combination of a collagen pedestal and micro collagen pillars containing cells expressing olfactory receptor. The collagen pedestal allowed to bring cells close air-liquid surface without getting dry damage. As a result, gaseous odorants detection was achieved by shortening diffusion length. Moreover, 3D structure of pillars enhanced reaction efficiency because of large surface. Therefore, we believe that our odorant detection system will be useful gaseous odorant sensors.

In this paper, we developed a centrifugal emulsification device toward high-throughput generation of monodisperse liposomes. The device generated lipid vesicles in two steps, membrane emulsification by asymmetric through-holes on a plate, and transfer of the emulsions to a planar monolayer at an oil/water interface. As a result of centrifugation of the device in a microtube, we succeeded in collection of cell-sized lipid vesicle. Since a number of the asymmetric through-holes can be fabricated on the plate by standard photolithographic process, and the reservoir for inner solution has relatively large volume (40 μl), the proposed device will be a powerful tool for massive generation of liposomes.

We propose a flexible glucose sensor combined with a parylene based electrode and a glucose-responsive fluorescent hydrogel for continuous monitoring of glucose concentration. By depositing parylene-C on a cupper (Cu) thin sheet and creating electrical circuits including a LED and a photo diode, we developed the flexible glucose sensor. The flexible glucose sensor could reduce damage to tissues and organs of patients because the flexibility allows its deformation following movements of the patients. Using the flexible glucose sensor, we succeeded in wireless measurement of glucose concentration in vivo by implantation of the sensor into a rat.

This research reports a method to fabricate a loop-shaped vessel-like channel for perfusion of culture medium in a cell-laden collagen gel. We used alginate fibers as sacrificial structures for construction of loop-shaped vessel-like channels. By integration of a loop-shaped alginate fiber with connection ports in a cell-laden collagen gel, we formed a tube-connectable loop-shaped channel. Finally, we seeded endothelial cells onto inner wall of the channel to fabricate vessel-like channel coated with endothelial cells. As a result, we achieved perfusion of culture medium via the loop-shaped vessel-like channel. We believe that this method will be useful for in vitro construction of three-dimensional (3D) tissues with complex-shaped vessel-like channels.

We developed a portable odorant detection system composed of collagen micro-pillars with cells expressing olfactory receptors and a CMOS imaging sensor; the CMOS imaging sensor caught fluorescence signals of Ca2+ increased by odorant molecules. While low sensitiveness of the CMOS imaging sensor was insufficient to detect the fluorescent signal from a single cell, the odorant detection system achieved supply of sufficient odorant-induced fluorescence intensity to the CMOS imaging sensor by vertical accumulation of the fluorescence from each cells in the pillars. We believe that our portable odorant detection system will be a useful platform for quantitative and selective portable odorant sensor.

We propose a microfluidic device for the supply of chemicals to selected parts of a meter-long cell-laden fiber (Fig.1). Because the fiber is fixed with anchors, we can make multiple laminar flows in the device without undesired movements of the fiber. The multiple laminar flows containing different chemicals allow us to selectively stimulate parts of the fiber. As a demonstration of the device function, we achieved labelling cells in selected parts of the fiber by providing a laminar flow with dye. We believe that the microfluidic device will be applicable to control localized cellular conditions in cell-laden fibers to investigate organogenesis and self-cure properties in vitro.

We propose a method to assemble fiber-shaped tissues into a 3D network. In the method, we connected fiber-shaped tissues using micro pillar devices anchoring both ends of the tissues. The fibershaped tissues were assembled into heterogeneous and 3D network. We believe that this method will be useful for the analysis of cellular morphology and functions in a 3D network of tissues.

We propose a fluorescence detection system for a portable odorant biosensor composed of cell spheroids with expressed olfactory receptors and a CMOS chip. As fluorescence intensity of spheroids is higher than that of single cells, we achieved detection of fluorescence signals caused by intracellular chemical reaction on the CMOS chip. As a demonstration of odorant detection, we prepared spheroids of cells expressing olfactory receptors to certain odorant molecules. The CMOS chip could detect the increment of the fluorescence intensity around the spheroids when odorant molecules were applied. We believe that the system will contribute to establish portable odorant biosensors.

This paper describes a method to form a branched hydrogel fiber that can be used as a sacrificial structure for vessel-like microchannel in 3D cell tissue. We established the method to fabricate a branched fiber using centrifugation and a V-cut θ-glass tube. We found that the configurations of fibers can be changed by controlling the centrifugal rotation speed and the θ-glass tube diameter. By embedding the fiber into collagen gel and dissolving it, we fabricated tissue model of cell-laden collagen gel integrated with branched channel. We believe that this technique can contribute to the field of tissue engineering.

We propose a drug testing system for quantifying contractile frequency and force of fiber-shaped tissues of human iPS-derived cardiomyocytes in various drugs. By optimization of cell culture conditions for the cardiomyocytes fiber and dimensions of the PDMS devices with cantilevers, WC succeeded in measurement of contractile frequency and force derived from deformations of the cantilevers. As a result of drug tests using our system, we achieved detection of changes of their contractile properties in accordance with physiological functions and reactions occurring in humans. Thus. WC believe that the system using the fiber of human iPS-derived cardiomyocytes will be a useful tool in first-in-human pharmacokinetic studies for drug development.

This conference proceeding describes a device for culturing skin-equivalent with vascular channels under dynamic stretching. The culture device is composed of a frame made of silicone rubber (polydimethylsiloxane (PDMS)/Ecoflex (R)) composite) and synthetic resin connections for tubing. Owing to the flexibility of silicone rubber, we can culture skin-equivalents not only perfusing medium but also applying stretching forces for various directions. Moreover, we applied a 1-axial cyclic force to the skin-equivalent during perfusion culture. As a result, we observed a thicker epidermis of the stretched skin equivalent than that of not-stretched one. Since the epidermis thickness is known as an important factor for the tensile strength of skin-equivalents, we believe that the device will enable the fabrication of robust skin equivalents with vascular channels useful for drugs/cosmetics testing, skin grafting and skin of biohybrid robots.

This paper reports a system enabling cell detection, fluorescence light detection and single cell retrieval. It consists of a device combining a microplate technology, allowing single cell seeding on individual cell-sized paiylene plates, and a CMOS imaging sensor placed on the right bottom of the microplate chip with glass substrate. By using such a system, we succeeded to visualize individual cells, to detect fluorescence light emission and to retrieve a single cell by using a micromanipulator. We believe that the proposed system will be useful for the point of care analysis on the level of single cell analysis.

© 15CBMS-0001. We developed a chemical vapor sensor inspired by the olfactory system. The sensor consists of an artificially configured membrane receptor between a buffer solution and agarose gel. The gel, mimicking mucus, enables to directly absorb target volatile molecules from vapor. The principle of the sensor is as follows: 1) DNA aptamer selectively captures the target molecules, and 2) builds up a molecular complex, 3) which inhibits the ionic current through a nanopore by blocking. We demonstrated the detection of omethoate molecule from vapor and defined a long-and-deep current blocking as the criterion of omethoate detection.

© 15CBMS-0001. We propose a system to place patterned three-dimensional (3D) skeletal muscle fibers on the bottom of a dish. In the system, fine images of shape-controlled muscle fibers can be taken using high-magnification lens since fabricated muscle fibers are proximity to the lens. Due to the fine observation, we were able to analyze cell nucleus density and width of single muscle fiber without any cut and manipulation. Moreover, we successfully analyzed single cell motion analyses on or in muscle fibers. From these results, we show that narrower muscle fibers realized more active motions of single myocyte. We believe that our system will be a useful tool for revealing the process of muscle formation in vitro.

© 15CBMS-0001. This paper reports a cell container composed of semipermeable membranes and a silicone rubber support for isolation of cells around environment. We can inject and store cells in the container without any leakage owing to flexibility of the silicone rubber. Moreover, the semipermeable membrane controls selective substance exchanges; blocking cells exchanges and allowing nutrition exchanges. We observed that viability of injected collagen-embedded cells in the container was as high as viability of collagen-embedded cells in a dish. We believe that the container will be useful for cell implantation since the container prevents immune cells from killing implanted cells.

This paper describes a vapor detecting system that applies two robust biological elements: A biological nanopore formed in a lipid bilayer and a DNA aptamer. The principle of the sensor is as follows: 1) DNA aptamer selectively captures the target molecules, 2) builds up a molecular complex larger than the nanopore size, and 3) inhibits the ionic current through the nanopore by blocking. We integrated these biological molecules into a previously developed device. A feasibility test was performed using a vapor phase sample, an organophosphorus pesticide, and represented the results demonstrating long-and-deep current blockades with the presence of the omethoate.

We propose a method for constructing fiber-type three-dimensional (3D) tissue of human iPS-derived cardiomyocytes and quantifying its contractile force in response to the addition of drug. By culturing the cardiomyocytes in micropatterned hydrogel with anchors, we succeeded in fabrication of the fibers with aligned cardiomyocytes and fixation of the fiber edges to the anchors. Since the fiber generated contractile force in a single direction due to alignment of cardiomyocytes, we can measure the contractile force accurately. Furthermore, as a demonstration of drug testing, we quantified contractile frequency and force in accordance with concentrations of pilsicainide. We believed that the fiber of human iPS-derived cardiomyocytes will be used in pharmacokinetic applications for drug development.

This paper describes a small sized balloon pump for providing liquid in low flow rate without a driving source. The balloon pump is composed of a balloon tank and a microfluidic valve. The balloon tank can work as a reservoir to store liquid and an actuator to pump liquid. By connecting the microfluidic valve to the balloon tank, we achieved extremely low flow rates of the liquid. Therefore, our balloon pump will be applicable to implantable pumps for the continuous drug supply in low flow rates without batteries.

We report a method to measure contractile forces of cardiomyocytes at cellular level using microplates; pairs of single-cell sized plates are connected by a flexible hinge at the center. Cardiomyocytes repeatedly contract and relax, and thereby fold and stretch the microplates at the flexible hinge. By measuring the change of the bending angle in folded microplates, we can estimate contractile forces of cardiomyocytes. We believe that this method is a useful tool to study the biomechanics of cardiomyocytes.

This conference proceeding describes a method to construct a skin-equivalent cultured on a curved surface. The skin-equivalent consists of not only the dermis/epidermis but also perfusable channels. We embedded an anchoring structure in a culture device to prevent the horizontal contraction of the tissue during cultivation. Owing to perfusion of culture medium via the channels, we can culture the epidermis at the air-liquid interface for cornification. This method enables the skin-equivalent construction on a curved surface; the curved surface is necessary for the construction of 3D complex skin surface and the covering of living skin on the 3D surface of biohybrid robots.

We propose a pneumatic balloon actuator capable of controlling its bending points. Local stiffness of the balloon actuator can be controlled by embedding low-melting-point-alloy that becomes solid by cooling. The bending points can be changed depending on the position of the melted low-melting-point-alloy since the pneumatic actuator bends at soft point. The advantage of our actuator is that its bending point can be simply controlled without changing the design of the whole device. We believe that the proposed actuation mechanism will be useful in designing highly flexible actuators for soft robotics.

© 15CBMS-0001. This conference proceeding describes a method to fabricate a skin-equivalent composed of epidermis and dermis integrated with perfusable vascular channels coated by human umbilical vein endothelial cells (HUVECs). By coating our device to construct a skin-equivalent having perfusable channels with O 2 plasma-treated parylene, the device was able to anchor a thicker skin-equivalent than previously reported. Moreover, by seeding HUVECs, the channels kept lumen structures while the channels without HUVECs were closed after incubation. According to these results, our method can be utilized for constructing a perfusable and vascularized skin-equivalent applicable to cosmetics testing or skin grafting.

© 14CBMS. This conference proceeding describes a balloon injector composed of a balloon actuator and a microfluidic regulator to achieve an implantable passive drug pump. The balloon actuator can work as a driving source to pump liquid. By connecting the leaky valve to the balloon actuator, we achieved smaller and more constant flow rate of the liquid. Therefore, our system will be applicable to implantable passive pumps for the constant supply of liquid with small flow rate without batteries.

© 14CBMS. We describe a centrifuge-based dynamic microarray system for trapping an array of single cells from small amount of suspension. We optimized microchannel designs and centrifugal force for minimizing sample volumes and maximizing trapping efficiencies, and achieved single-cell trapping by introducing only 5 μl of cell suspension with the centrifugal force of 70 G. Trapping of cells stained with DAPI shows that single cells were trapped at desired trapping spots. This system will be applicable for single cell assays using rare or expensive cells.

© 14CBMS. This conference proceeding describes a semipermeable poly-L-lysine (PLL) tube toward construction of a vascular-like channel in 3D cell culture. By shaping a PLL-coated alginate gel fiber and removing the alginate from the fiber in collagen gel, we fabricated an arbitrarily-shaped channel with a PLL-tube. Even when mechanical stress was applied, the channel kept its shape due to stiffness of the PLL-tube. In order to show the feasibility for perfusion, we confirmed that the channel was hollow by observing flow of micro-beads. Accordingly, we believe that our channel is useful as a nutrition pathway for long-term culture of 3D tissue construct.

© 14CBMS. Brain stereotaxic surgery is a useful tool in system neuroscience. However, the surgery in mouse pups has difficulties caused by anesthetic control and brain stereotaxis. Here, we develop an inhalation anesthetic device equipped with brain stereotaxic function specialized for mouse pups. We successfully anesthetized a pup and performed brain microinjection at once using this single device. The pup recovered quickly when it was removed from the device. Since the channel fabrication process is compatible to integrate with various fluidic components, our approach will provide a handy and functional instrument for stereotaxic surgery in the developing brain.

© 14CBMS. We propose a muscle actuator with tendon-like structures to mimic configurations of our body. In the muscle actuator, two muscles are arranged in plane symmetry to a link mechanism, and tendon-like structures are located between the edges of the muscles and the link mechanism. Because deformation of the tendon-like structures prevent the muscles from deforming, the link mechanism can rotate smoothly. Moreover, selective contraction of each muscle allowed the actuator to drive bidirectionally. From tetanic contraction results, we show that the strain of contracted muscle is about 20%, similar to that of our body. We believe that the muscle actuator is useful not only as an in vivo-like actuator in biorobotics field, but also for revealing biological properties of muscles in our body.

This chapter introduces the reproducible fabrication methods of cell-laden hydrogel modules with a controllable design and the characteristics of the modules. It provides an overview of handling techniques of the modules in microfluidic devices. The chapter talks about applications of the modules for transplantation and bottom-up tissue engineering. Combined with cell assay microfluidic systems and cell-laden hydrogel modules, microtissues with extracellular matrices (ECMs) are useful for analyses of cell functions and cell-cell interactions because microtissues can easily be handled, arrayed, and retrieved in microfluidic systems. Furthermore, the cell-laden hydrogel modules can be used as building units for reconstructing 3D cell structures. Therefore, the cell-laden hydrogel modules produced by microfluidic devices have a great potential to create miniaturized tissues for human implantation and for treatment of diseases. © 2013 by The Institute of Electrical and Electronics Engineers, Inc.

We propose a muscle actuator with antagonist muscle to mimic configurations of our body. In the muscle actuator, two bundles of muscle fibers are arranged in plane symmetry, and a flexible substrate with electrodes is located at the center of the two muscle bundles. Owing to the existence of the antagonist muscle, tension of the two muscle bundles balanced with each other during culture. Moreover, selective contractions of each muscle bundle allowed the substrate to bend bidirectionally. From the contraction results, we were able to estimate a contractile force of muscle and a reaction force of antagonist muscle. We believe that the muscle actuator is useful not only as an in vivo-like actuator in biorobotics field, but also for revealing biological properties of muscle with antagonist muscle. Copyright © (2013) by the Chemical and Biological Microsystems Society All rights reserved. All rights reserved.

This paper describes a rapid bone tissue fabrication using chemically modified collagen microbeads. We prepared a collagen, activated photo-crosslinkable hyaluronan (API I), aminosilane and aminophosphate mixture, and then formed hydrogel microbeads by using axisymmetric flow focusing devices (AFFD) [1] . Phosphate or silane-modified surface would easily bind with inorganic molecules, and thus hydroxyapatite (IIA) was easily crystallized. Rat newborn primary osteoblasts (RNOs) were cultured onto microbeads and successfully calcified within 3 days. Bone remodeling takes long term ( > 28 days) because it is difficult to enlarge the number of cells adhering to scaffolds with keeping its mechanical strength. Remodeling speed would be accelerated by accumulating calcified osteo-beads because of their rapid growing speed of IIA. Therefore, we believe that this method would be useful for rapid preparation of bone grafts. Copyright © (2013) by the Chemical and Biological Microsystems Society All rights reserved. All rights reserved.

This report describes preliminary results of reconstitution of an in vivo-like multi-layered placental barrier without porous membrane support. We used thin collagen membrane (10 mu m in thickness, commercially available) as permeable support of placental barrier structure and cultured BeWo trophoblastic cells and HUVECs on either side of the membrane. Co-cultured cells deposited abundant basement membrane matrices that support cellular function, whereas the deposition was hardly observed on polymer-based porous membrane conventionally used in organ-on-chip study. These results suggest that the placental barrier-like multi-layered construct integrated with microfluidic channels may serve as a platform for transplacental nutrients transport and pharmacokinetics studies.

We propose a muscle based bioactuator that can be driven in air. As the driving force of this actuator, we mounted aligned muscle fiber sheets between poles, with gold electrodes located at the edges of the muscle fibers to stimulate them. The muscle fibers are in a hollow space covered by a collagen structure to preserve the actuating properties in a wet condition. In the experiments, we prove that the muscle fiber sheets with striped patterns increase the forces by improving the alignment of muscle fibers, and the hollow space facilitates the contraction of muscle fibers smoothly. Owing to the collagen encapsulation, the muscle fibers were able to contract in wet condition even if the whole structure is in air; thus this bioactuator can bend in air. We believe that the bioactuator can be a useful module to construct soft robots as well as engineered tissue models for muscular cell biology and drug development.

This paper describes a tunable lenticular lens to switch high-resolution two-dimensional (2D) / three-dimensional (3D) images in naked-eye stereoscopic displays. This lens is simply composed of transparent poly(dimethylsiloxane) (PDMS) microchannels and pumps to realizes two states: (A) flat surface for light transmissions without deflection
(B) cylindrically deformed surface for light deflection. This lens has three advantages: (i) switchable optical characteristics without patterned electrode, (ii) adjustable focal length by the pressure-driven deformation, (iii) simple fabrication and low-cost experimental setup. By using this device put on a Smart Phone display, we successfully observed 3D images and two different images depending on view angle. Moreover, we obtained clear 2D images without decrease in the horizontal resolution. We believe that this device can be applied to a portable and high luminance naked-eye stereoscopic display by the combination with various types of commercially available portable displays. © 2013 IEEE.

We propose a method for constructing "neuron-muscle fibers (NM fibers)" driven by neural signals from activated neurons. The NM fibers consist of stretched muscle fibers covered with aligned neurons, and the neurons extend their axons into the muscle fibers and form neuromuscular junctions (NMJs). We successfully constructed the neuron-muscle fibers by culturing neural stem cells on stretched muscle fibers; the neural stem cells then differentiated into neurons and connected to the muscle cells, forming NMJs. Furthermore, we found that the neuron-muscle fibers contract after treatment of glutamic acid that activates neurons to stimulate the muscle cells. We believe that our neuron-muscle fibers will be not only used as soft-robotic actuators controlled by neuronal signals, but also used in therapeutic and pharmacokinetic assays for NMJ disease models.

We propose a method to construct a biohybrid tissue-like structure. In this structure, muscle fibers are fixed at several poles and maintain their tensions, and gold electrodes are located at the edges of the muscle fibers. Therefore, we can exercise contractions of the specific muscle fibers via the electrodes. Since a device with muscle fibers and electrodes is free-standing, it can be integrated in three-dimensional (3D) in vivo-like environments composed of living cells and extracellular matrixes (ECMs). We believe that our method is useful for fabrication of 3D hierarchic muscular structures for the evaluations of muscle behaviors in in vivo-like conditions.

We propose a method for constructing "neuron-muscle fibers" driven by signals from activated neurons. The fibers consist of stretched muscle fibers covered with highly aligned neurons, and the axons of neurons extend into muscle fibers and form neuromuscular junctions (NMJs). We successfully constructed the neuron-muscle fibers by culturing neural stem cells on stretched muscle fibers; the neural stem cells then differentiated into neurons and connected to muscle cells, forming NMJs. Furthermore, we found that the neuron-muscle fibers contract after treatment of glutamic acid that activates neurons to stimulate the muscle cells. We believe that our neuron-muscle fibers will be not only used as soft-robotic actuators controlled by neuronal signals, but also used in therapeutic and pharmacokinetic assays for NMJ disease models.

This paper describes a method of three-dimensional (3D) neuronal pathway formation in vitro by bottom-up neuronal tissue block assembly (Fig.1). We first formed the neuronal tissue blocks by molding uniform-sized neurospheroids into the PDMS mold chambers. We then assembled a millimeter-sized cortex-hippocampal tissue by connecting cortical and hippo-campal tissue blocks. We found that cortical and hippocampal neurons extended their axons to each other, and that 3D neuronal pathway was successfully formed in the 3D environment. We believe that our neuronal block assembly enables the fabrication of in vivo-like neuronal pathway, and that this system is useful tool for neuronal tissue engineering, pharmacological assay for the screening of drugs and toxins to neuronal pathway.

We propose aligned free-standing skeletal muscle fibers connecting with neurons. By confining skeletal cells, C2C12, with collagen gel into polydimethylsiloxane (PDMS) trenches, we successfully formed the muscle fibers that are highly oriented to the fiber direction. We cultured the free-standing muscle fibers maintaining their tension since the edge of the fibers was fixed to glass substrates. Furthermore, we can culture the muscle fibers with rat primary neurons, and the neurons successfully connect with the muscle fibers. We believe that the muscle fibers connected with the neurons have a potential to contract by the nerve impulse. Copyright © (2011) by the Chemical and Biological Microsystems Society.

We successfully established a three-dimensional (3D) hierarchic cell co-culture system using micro-collagen beads. Our reconstructed tissues have double cell-layers that provide an in vivo-like micro-environment. To prepare the hierarchic tissue structures, we used an axisymmetric flow-focusing device (AFFD) that allows us to encapsulate HepG2 cells within monodisperse collagen beads. We then seeded 3T3 cells on the surface of the collagen beads. We observed that HepG2 cells and 3T3 cells successfully self-organized into hierarchical cell structures of uniform size. We believe the in vitro 3D co-culture beads are useful for the studies of in vivo-like cell-cell interactions with various cells. Moreover, the monodispersity of the 3D cell cultured beads facilitates on-chip assays of chemicals/drugs.

We describe a three-dimensional (3D) cell culture system using monodisperse gel beads. Cells were non-invasively encapsulated in biocompatible hydrogels by using a 3D microfluidic axisymmetric flow-focusing device. This technique holds great promise for the further study of fundamental cell-cell communication and mechanism in 3D cultured cells, and reconstruction of viable tissues for the regenerative medicine.

We describe an inexpensive technique for rapidly generating microfluidic devices. We use a cutting-plotter machine to cut commercially available watersoluble starch sheets into channels to build the master mold for the devices. We degas the poly(dimethylsiloxane) (PDMS) with a portable vacuum sealer and cure it in a microwave. Using this method, we can fabricate T-junction microfluidic devices, stamps for micro-contact printing and 3D microfluidic channels. The total processing time is less than 90 min. This method can be used by scientists without access to microfabrication facilities and can be applied in science education. © 2008 CBMS.

We describe a three-dimensional (3D) cell culture system using monodisperse peptide hydrogel beads. We utilized a self-assembling peptide hydrogel to provide cells in vivo-like microenvironment. We succeeded in encapsulating endothelial cells within the hydrogels by using a 3D microfluidic axisymmetric flow-focusing device (AFFD), and showed the encapsulated endothelial cells were viable and were able to migrate within the gels.

We fabricated a hybrid axisymmetric flow-focusing device (h-AFFD) by combining photolithography and stereolithography for producing monodisperse microsized-emulsions (~Ø12 μm). We realized three-dimensional and highresolution structures in the h-AFFD, these structures can not be embedded using either stereolithography or photolithography alone. While the h-AFFD still maintains the same hydrodynamic performance as the monolithic AFFD, the h- AFFD can produce the smaller droplets than the monolithic one due to its narrow orifice (~Ø50 μm) fabricated by photolithography. In addition, as the wetting problem does not occur in the h-AFFD, we succeeded in producing monodisperse emulsions containing single cells without surface modifications. © 2008 CBMS.

We present a method to form monodisperse microcages encapsulating cells with poly-L-lysine (PLL) membrane. These microcages were prepared with a monolithic three-dimensional microfluidic axisymmetric flow-focusing device (3D AFFD) using internal gelation method. The production process of microcage is mild enough for cells. The microcages were sufficiently monodisperse and robust to be trapped in a beads-based microfluidic array system for easy observation. We also confirmed that (i) the PLL membrane is semi-permeable, (ii) cells are able to move freely inside the microcages, and (iii) cells can be successfully cultured inside these microcages.