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A highly sensitive odorant sensor was developed by using insect olfactory receptors reconstituted into a lipid bilayer membrane., This paper reports a volatile organic compound (VOC) sensor based on olfactory receptors that were reconstituted into a lipid bilayer and used in a specifically designed gas flow system for rapid parts per billion (ppb)–level detection. This VOC sensor achieves both rapid detection and high detection probability because of its gas flow system and array design. Specifically, the gas flow system includes microchannels and hydrophobic microslits, which facilitate both the introduction of gas into the droplet and droplet mixing. We installed this system into a parallel lipid bilayer device and subsequently demonstrated parts per billion–level (0.5 ppb) detection of 1-octen-3-ol in human breath. Therefore, this system extends the various applications of biological odorant sensing, including breath diagnosis systems and environmental monitoring.

An artificial cell membrane is applied to study the pore formation mechanisms of bacterial pore-forming toxins for therapeutic applications. Electrical monitoring of ionic current across the membrane provides information on the pore formation process of toxins at the single pore level, as well as the pore characteristics such as dimensions and ionic selectivity. However, the efficiency of pore formation detection largely depends on the encounter probability of toxin to the membrane and the fragility of the membrane. This study presents a bilayer lipid membrane array that parallelizes 4 or 16 sets of sensing elements composed of pairs of a membrane and a series electrical resistor. The series resistor prevents current overflow attributed to membrane rupture, and enables current monitoring of the parallelized membranes with a single detector. The array system shortens detection time of a pore-forming protein and improves temporal stability. The current signature represents the states of pore formation and rupture at respective membranes. The developed system will help in understanding the toxic activity of pore-forming toxins.

Summary Hydrogel glucose sensors with boronic acid-based fluorescence intensity theoretically hold promise to improve in vivo continuous glucose monitoring (CGM) by facilitating long-lasting accuracy. However, these sensors generally degrade after implantation and the fluorescence intensity decreases immediately over time. Herein, we describe a hydrogel glucose sensor with in vivo stability based on boronic acid-based fluorescence intensity, integrating two antioxidant enzymes, superoxide dismutase (SOD), and catalase. These protected the arylboronic acid from being degraded by hydrogen peroxide in vitro and preserved the boronic acid-based fluorescence intensity of the hydrogel glucose sensors in rats for 28 days. These antioxidant enzymes also allowed the hydrogel glucose sensor attached to a homemade semi-implantable CGM device to trace blood glucose concentrations in rats for 5 h with the accuracy required for clinical settings. Hydrogel glucose sensors with boronic acid-based fluorescence intensity containing SOD and catalase could comprise a new strategy for in vivo CGM., Graphical Abstract, Highlights • The arylboronic acids of hydrogel glucose sensors are sensitive to cleavage by ROS • The antioxidant enzymes suppress the degradation of fluorescence effectively in vivo • The developed sensor performs CGM with the accuracy required for clinical settings, Biochemical Mechanism; Glycobiology; Medical Device; Sensor

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...

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

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.

MicroRNAs have critical roles in a number of serious diseases and, as a result, are of major interest as clinical diagnostic targets. Conventionally, microRNAs are collected from blood and urine samples and are measured by either quantitative reverse-transcription polymerase chain reaction or microarray. Recently, nanopore sensing techniques have been applied for measuring microRNAs at the single-molecule level. However, existing techniques are technically complex, needing several tools and requiring purification and/or labeling of microRNA samples prior to use. Here we report a method for microRNA detection in a simple procedure requiring neither purification nor labeling. This system utilizes magnetic beads anchored with DNA and nanopores on a liposome membrane. In the presence of the target microRNA, it forms a duplex with complementary DNA, which is then cleaved by a duplex-specific nuclease (DSN). The cleaved DNA, which harbors a liposome on its terminus, is subsequently released from the magnetic bead, fuses to the lipid bilayer on chip, and emits an electrical signal derived from the formation of a nanopore. Because of a property of the DSN, the signals derived from microRNAs are expected to be amplified in an isothermal reaction. Our system possesses the specificity to detect target microRNAs from mixtures containing >106 different microRNA sequences and readily uses blood or urine samples. Although the limit of detection is above 10 nM and needs to be improved for practical diagnosis, because purification and labeling are not required, the presented system proposes a possible schematic for the development of easy and on-site diagnosis.

Current methods of generating asymmetric lipid vesicles produce only single types of vesicles, which poses a challenge for investigation of vesicles with different lipid leaflet combinations using a single device. Here, we describe a device for sequentially generating asymmetric lipid giant vesicles (GVs) with various combinations of asymmetric lipid leaflets. Various combinations of planar asymmetric lipid bilayers are formed by sliding and contacting the water in oil (phospholipid) (W/O) droplets in the collecting and jetting wells of our device. Next, we generate asymmetric lipid vesicles using a pulsed-jetting method. We sequentially generate three types of GVs: two asymmetric GVs containing fluorescent-conjugated phospholipids (either (i) rhodamine or (ii) BODIPY) on the outer leaflet, and (iii) a symmetric GV with phosphatidylcholine (PC) on the inner and outer leaflets. The use of asymmetric GVs with various combinations of asymmetric lipid leaflets reveals that increase in membrane phosphatidylethanolamine (PE) concentration influences cinnamycin activity and promotes the phospholipid flip-flop dynamics. This system will be useful for investigating activities of proteins or peptides on GV membranes with various combinations of lipid leaflets.

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

Transplantation technologies of pancreatic islets as well as stem cell-derived pancreatic beta cells encapsulated in hydrogel for the induction of immunoprotection could advance to treat type 1 diabetes mellitus, if the hydrogel transplants acquire retrievability through mitigating foreign body reactions after transplantation. Here, we demonstrate that the diameter of the fiber-shaped hydrogel transplants determines both in vivo cellular deposition onto themselves and their retrievability. Specifically, we found that the in vivo cellular deposition is significantly mitigated when the diameter is 1.0 mm and larger, and that 1.0 mm-thick xenoislet-laden fiber-shaped hydrogel transplants can be retrieved after being placed in the intraperitoneal cavities of immunocompetent diabetic mice for more than 100 days, during which period the hydrogel transplants can normalize the blood glucose concentrations of the mice. These findings could provide an innovative concept of a transplant that would promote the clinical application of stem cell-derived functional cells through improving their in vivo efficacy and safety.

Lipid distribution in intracellular vesicles is different from that in the plasma membrane of eukaryotic cells. The lipid components in the intracellular vesicles are composed of phosphatidylserine and phosphatidylethanolamine in the outer leaflet and phosphatidylcholine and sphingomyelin in the inner leaflet. The lipid asymmetricities both in the intracellular vesicle membrane and the plasma membrane contribute to synaptic transmission functions. In this study, we developed a cell-sized asymmetric lipid vesicle system containing small-sized asymmetric lipid vesicles (of diameter 200–1000 nm) (asymmetric vesicles-in-a-vesicle), emulating lipid components in the plasma membrane and intracellular vesicle membrane of eukaryotic cells, using microfluidic technology. We successfully constructed an artificial exocytosis system using the asymmetric vesicles-in-a-vesicle system. This asymmetric vesicles-in-a-vesicle system will be helpful in understanding the mechanisms of vesicle transport, such as neurotransmission and exocytosis.

In this paper, we present a microfluidic device for electrofusion of liposomes. The device contains hydrodynamic microarray system for pairing liposomes and two microelectrodes for the application of electric field on the paired liposomes. Arraying multiple pairing channels, we demonstrated the electrofusion of paired liposomes in the dynamic microarray structure. We believe this device will become a useful tool for analysis of biochemical reaction inside liposomes and for introducing external substances into cells.

Voltage-clamp fluorometry (VCF) has been combined with artificial lipid bilayer systems to optically measure the dynamics and contribution of membrane proteins on molecular transport at the clamped membrane potential. However, the previous methods for VCF require expensive apparatus, such as total internal reflection fluorescence microscope, or require extensive experience for voltage-clamping, or possibly lead to imprecise membrane potential. This study describes VCF performed using a pico-liter-sized lipid bilayer chamber with an embedded Ag/AgCl microelectrode. Owing to the size of the picoliter order, molecular transport through the lipid bilayer can be detected using a normal fluorescent microscope. Easy electrical access into the lipid bilayer chamber can be realized because the microelectrode is spontaneously embedded inside when forming the lipid bilayer chamber. The microelectrode is made of Ag/AgCl; therefore, it does not tend to polarize, which results in reliable regulation of the membrane potential. Using the developed system, we performed the VCF of molecular transport through the lipid bilayer by incorporating the pore-forming membrane protein, α-hemolysin. This proposed method will increase the number of researches that can perform the VCF of molecular transport through artificial lipid bilayers.

Most organs contain interconnected tubular tissues that are one-cell-thick, polarized epithelial monolayers enclosing a fluid-filled lumen. Such tissue organization plays crucial roles in developmental and normal physiology, and the proper functioning of these tissues depends on their regulation by complex biochemical perturbations and equally important, but poorly understood, mechanical perturbations. In this study, by combining micropatterning techniques and atomic force microscopy, we developed a simple in vitro experimental platform for characterizing the mechanical properties of the MDCK II cyst, the simplest model of lumen-enclosing epithelial monolayers. By using this platform, we estimated the elasticity of the cyst monolayer and showed that the presence of a luminal space influences cyst mechanics substantially, which could be attributed to polarization and tissue-level coordination. More interestingly, the results from force-relaxation experiments showed that the cysts also displayed tissue-level poroelastic characteristics that differed slightly from those of single cells. Our study provides the first quantitative findings, to our knowledge, on the tissue-level mechanics of well-polarized epithelial cysts and offers new insights into the interplay between cyst mechanics and cyst physiology. Moreover, our simple platform is a potentially useful tool for enhancing the current understanding of cyst mechanics in health and disease.

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.

In this paper, a tubular 3D microenvironment created in a calcium alginate hydrogel microtube with respect to the effect of scaffold dimensions on the differentiation of mouse neuronal stem cells (mNSCs) is evaluated. Five types of hydrogel microtubes with different core diameters (≈65-200 μm) and shell thicknesses (≈30-110 μm) are fabricated by using a double coaxial microfluidic device, and differentiation of encapsulated mNSCs is induced by changing the growth medium to the differentiation medium. The influence of the microtube geometries is examined by using quantitative real-time polymerase chain reaction and fluorescent immunocytochemistry. The analyses reveal that differences in microtube thickness within 30-110 μm affected the relative Tuj1 expression but do not affect the morphology of encapsulated mNSCs. The diameters of cores influence both the relative Tuj1 expression and morphology of the differentiated neurons. It is found that the tubular microenvironment with a core diameter of less than ≈100 μm contributes to forming highly viable and aligned neural tissue. The tubular microenvironment can provide an effective method for constructing microfiber-shaped neural tissues with geometrically controlled differentiation induction.

Nano-sized lipid vesicles (50–300 nm in diameter) are widely used in drug delivery systems and cosmetics. Recently, exosomes have been shown to be important as the carriers of biomolecules. The membrane structures of exosomes exhibit the same asymmetric lipid distributions as those of the plasma membranes of eukaryotic cells. The asymmetric lipid distributions of the exosome membranes play a key role in the interactions with and recognition of living cells. The asymmetric lipid distributions of nano-sized lipid vesicles facilitate delivery in advanced drug delivery systems, which emulate the cellular transportation systems of exosomes. In this study, we generate nano-sized asymmetric lipid vesicles using a pulsed-jet method for cell-sized asymmetric lipid vesicle formation. They were generated from an asymmetric planar lipid bilayer by applying pulsed-jet flows of longer duration and higher pressure than those used in the formation of micro-sized lipid vesicles. Using a transmission electron microscope, nano-sized lipid vesicles with thicknesses of approximately 5–6 nm (approximately 70 % of the generated sample), unilamellarity, and asymmetry were observed to be produced by this method. The nano-sized asymmetric lipid vesicles using this method have the potential to be useful as carriers in drug delivery systems.

In this paper, we developed a spheroid culture device that can trap a spheroid in the trapping site sandwiched by two extracellular matrix gels located at the upper and lower side of the spheroid. This device can form different biochemical gradients by applying target biochemicals separately in upper and lower channels, allowing us to study the angiogenic sprouting under various biochemical gradients in different directions. In the experiments, we confirmed the trapping of the spheroids and demonstrate the investigation on the direction and extent of angiogenic sprouts under unidirectional or bidirectional biochemical gradients. We believe our device can contribute to understanding the pathophysiological phenomena driven by chemical gradients, such as tissue development and tumor angiogenesis.

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

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.

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.

Ion channels are located in plasma membranes as well as on mitochondrial, lysosomal, and endoplasmic reticulum membranes. They play a critical role in physiology and drug targeting. It is particularly challenging to measure the current mediated by ion channels in the lysosomal and the endoplasmic reticulum membranes using the conventional patch clamp method. In this study, we show that our proposed device is applicable for an electrophysiological measurement of various types of ion channel in plasma and organelle membranes. We designed an on-chip device that can form multiple electrical contacts with a measurement system when placed on a mount system. Using crude cell membranes containing ion channels extracted from cultured cells without detergents, we detected open/close signals of the hERG, TRPV1, and NMDA channels on plasma membranes, those of the TRPML1 channels on lysosomal membranes, and open/close signals of the RyR channels on SR membranes. This method will provide a highly versatile drug screening system for ion channels expressed by various cell membranes, including plasma, SR, mitochondrial, Golgi, and lysosomal membranes.

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.

Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of “multi-cellular engineered living systems,” which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered.

This paper describes automation of planar lipid bilayer formation by introducing a stepping motor to a microfluidic device. Planar lipid bilayers or lipid vesicles are useful to understand biological reactions and to investigate the interaction between lipids and proteins. Therefore, to acquire large amount of the information, high-throughput production of planar lipid bilayers or giant vesicles (GVs) is necessary. The droplet split-and-contact method, which enhances the efficiencies of both planar lipid bilayer formation and GV generation, needs to be automated for increasing the throughput. Previous droplet split-and-contact devices were manipulated manually; hence, the influence of manipulation on planar lipid bilayer formation was not evaluated quantitatively. First, to develop an automated system for generating asymmetric planar lipid bilayers, a stepping motor, which allows to control the angular speed of the rotor, is integrated into the droplet split- and-contact device (Fig. $1(\mathrm{b)$). Next, we assessed planar lipid bilayer generation at various angular speeds and found the speed limit for bilayer formation. Finally, we generated asymmetric planar lipid bilayers that have different lipid composition on outer and inner leaflets using this automated device and confirmed the asymmetry of the planar lipid bilayers by generating GVs.

This article describes a three-dimensional culture system for the expansion of anchorage-dependent cells using fiber-shaped microcarrier (MC; Cytodex3) aggregates, termed "MC fibers." The fiber encapsulates the cells, the MC aggregates, and collagen and is covered with a poly-l-lysine membrane. The thin structure of the fiber enables sufficient supply of O2 and nutrients to the cell. Using the MC fiber, we demonstrated the efficient expansion of C2C12 cells with high viability through serial passaging. Therefore, our culture system is useful for various applications where large-scale cell expansion is required, such as in pharmaceutical technologies, regenerative medicine, and cultured meat production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2755, 2019.

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.

This manuscript describes selective pairing and fusion of biological vesicles using dielectrophoretic (DEP) tweezer. Fusion of biological vesicles have been widely studied for artificial cell system, however, low selectivity of fused vesicle has been problematic. We propose selective pairing and fusion of vesicles such as cell-sized liposomes and giant bacteria using microfabricated DEP tweezer. As DEP force can manipulate cell-sized (approximately 10 μm) objects, for the first time we achieved selective pairing and fusion of the vesicles and in conjunction with electrofusion device. We believe our method will be useful in vesicle fusion studies including material transport and generation of artificial cells.

This paper describes a multicore-shell hydrogel fiber encapsulating multi type cell with lotus root structure (Fig. 1). Microfluidic techniques are able to encapsulate the cells in 3D configuration such as beads and fibers. Especially, an excellent method was reported that the core-shell cell microfibers reconstitute intrinsic morphologies and functions of living tissues in vitro [1]. Among various tissues and organs that have been targeted for in vitro reconstruction using cell microfiber application, various types of cells, such as pancreatic β cells [2], adipocytes [3], neural cells [4] and stem cells [5], can be cultured in a 3D tubular hydrogel microenvironment. However, cell distribution in vivo is extremely complex, and spatially anisotropic or heterogeneous tissues are widespread. We here took an approach to fabricate multicore-shell fibers with controlled heterogeneous cell-laden structures for construction of macro-size tissue. The fibers mimicked the structure of lotus root and the encapsulated cells in the fiber separately cultured as multi cell type co-culture system.

This paper describes a method of sequential formation of asymmetric lipid vesicles, which applies pulse jet flow to an asymmetric planar lipid bilayer with multiple lipid components formed by Droplet Split-and-Contact Method. In our previous work, we generated asymmetric lipid vesicles by deforming an asymmetric planar lipid bilayer using a pulsed jet flow. However, it was difficult to create various types or large amounts of the asymmetric lipid vesicles in a single double-well device. In this study, asymmetric planar lipid bilayers with multiple lipid components were formed by contacting various types of lipid monolayers using movable wells on a revolving table. We successfully produced two types of the asymmetric lipid vesicles containing red or green fluorescent lipids on the outer leaflet with a single device in a high-throughput manner using the proposed method. The asymmetricity of the generated vesicles was evaluated by the distribution of the fluorescence intensities. The proposed device will be applicable for generating artificial cell models with multiple asymmetric lipid components.

Multicellular spheroids are indispensable in cell biology as three-dimensional environments, in tissue engineering for achieving higher functions, and in drug validation by pharmaceutical companies. However, spheroids have limited nutrient exchange because they lack microvasculature. In this study, we established a method for fabricating microchannel networks in multicellular spheroids to overcome this limitation. Alginate hydrogel beads with almost the same size as animal cells were prepared by discharging alginate solution using an inkjet system. Using these beads, we produced “heterospheroids” that comprised the same number of cells and hydrogel beads based on the rapid cell/particle aggregation method. The hydrogel beads occupied the branched, connected spaces in the heterospheroids, which resembled microvasculature. The connectivity of the microchannel networks was confirmed using confocal laser microscopy and staining cells with 1-μm polystyrene beads after digesting the hydrogel beads using alginate lyase solution. The microchannel networks improved the albumin secretion rate and suppressed the expression of hypoxia-inducible factor-1α in Hep G2 cells. Experiments with rat primary hepatocytes demonstrated that the branched luminal-like structures overcame the limitations of albumin secretion and ammonia clearance. These findings suggest that it is possible to fabricate microchannel networks that can effectively maintain cellular functions by enhancing material exchange.

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.

Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core-shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.

In bottom-up tissue engineering, a method to integrate a pathway of nutrition and oxygen into the resulting macroscopic tissue has been highly desired, but yet to be established. This paper presents a cellular building unit made from microstrand-shaped bacterial cellulose (BC microstrand) covered with mammalian cells. The BC microstrands are fabricated by encapsulating Acetobacter xylinum with a calcium alginate hydrogel microtube using a double co-axial microfluidic device. The mechanical strength and porous property of the BC microstrands can be regulated by changing the initial density of the bacteria. By folding or reeling the building unit, we demonstrated the multiple shapes of millimeter-scale cellular constructs such as coiled and ball-of-yarn-shaped structures. Histological analysis of the cellular constructs indicated that the BC microstrand served as a pathway of nutrition and oxygen to feed the cells in the central region. These findings suggest that our approach facilitates creating functional macroscopic tissue used in various fields such as drug screening, wound healing, and plastic surgery.

MicroRNA, a short-length RNA known as a biomarker of cancers, was detected by magnet-attached spiral channel and electrical measurement. A designed oligo-DNA bound with magnetic bead was entrapped in spiral channel by magnetic field, and hybridized with microRNA. Thereafter, liposome were exported from the spiral channel by duplex-specific nuclease, and applied to electrical measurement. Finally, ionic current signals were observed specifically when the target microRNA was applied. Whereas, when a mixture of over 106 different mock microRNAs were applied, no signals were observed. This indicates a noise-free, high selectivity was achieved in the presented device.

This work presents a method to detect volatile odorant using corneal epithelial cells with olfactory receptors. Because the corneal epithelial cells can survive at constant exposure of air, these cells have ability to detect the volatile odorant at air-liquid phase in contrast to other parenchymal cells. Therefore, in the present study, we first fabricate a device that provides the air-liquid environment by (i) a dorm shaped chamber to confine the air and (ii) channels to maintain humidity of the cells on the bottom of the device. Using the device, we successfully detected the volatile odorant using the fluorescence imaging of the corneal epithelial cells that were transfected with the mammalian olfactory receptors.

This paper proposes a formation method for oil-layer-free bilayer lipid membranes (BLM) by using a compressive force of DC electric field. Using a droplet contact device previously developed, constant-voltage steps were applied to the pre-bilayer membrane and carefully controlled the electric stress in response to the membrane features, based on optical and current monitoring. We examined the pathways of the membrane formation and disruption, and discovered a qualitative protocol for oil-layer-free BLM formation. We succeeded in BLM formation using 1:1-mixture of hexadecane and n-decane as an oil example, which was hardly available for the former studies using the device. The method would contribute to produce vivo-like membranes appropriate for membrane protein functionalities.

A collagen sheet with highly aligned collagen fibers is fabricated by continuous cyclic stretch. The rearrangement of the collagen fibers depends on the different process parameters of the cyclic stretch, including magnitude, frequency, and period of stretch. The collagen fibers are aligned perpendicularly to the direction of the stretch. Corneal stromal cells and smooth muscle cells cultivated on the highly aligned collagen sheet show alignment along the collagen fibers without the stretch during culture. Thus, the sheet can be a suitable scaffold for use in regenerative medicine.

This paper describes a methodology for the rapid and highly selective detection of cocaine using a membrane protein channel combined with a DNA aptamer. The DNA aptamer recognizes the cocaine molecule with high selectivity. We successfully detected a low concentration of cocaine (300 ng/mL, the drug test cutoff limit) within 60 s using a biological nanopore embedded in a microchip.

Neural transplantation therapy using neural stem cells has received as potential treatments for neurodegenerative diseases. Indeed, this therapy is thought to be effective for replacement of degenerating neurons in restricted anatomical region. However, because injected neural stem cells integrate randomly into the host neural network, another approach is needed to establish a neural pathway between selective areas of the brain or treat widespread degeneration across multiple brain regions. One of the promising approaches might be a therapy using pre-made neural network in vitro by the tissue engineering technique. In this study, we engineered a three-dimensional (3D) tissue with a neuronal network that can be easily manipulated and transplanted onto the host brain tissue in vivo. A polydimethylsiloxane microchamber array facilitated the formation of multiple neurospheroids, which in turn interconnected via neuronal processes to form a centimeter-sized neurospheroid network (NSN). The NSN was transferable onto the cortical surface of the brain without damage of the neuronal network. After transfer onto the cortical tissue, the NSN showed neural activity for more than 8 days. Moreover, neurons of the transplanted NSN extended their axons into the host cortical tissue and established synaptic connections with host neurons. Our findings suggest that this method could lay the foundation for treating severe degenerative brain disease.

In this study, we established rat primary hepatocyte sandwich cultures on oxygen-permeable membranes and investigated the change in their repolarization. Functional bile canaliculi in sandwich-cultured hepatocytes on oxygen-permeable polydimethylsiloxane (PDMS) membranes were re-established more quickly than those in a conventional sandwich culture on polystyrene (PS). This enhanced biliary excretory activity was also observed in hepatocytes on another oxygen-permeable membrane plate but not on a PDMS surface whose oxygen permeability is blocked. An apical efflux transporter protein, Mrp2, was more rapidly distributed in hepatocytes cultured on PDMS membranes than in hepatocytes cultured on conventional PS plates. Moreover, the area of distribution of the Mrp2 in polarized hepatocytes cultured on PDMS membranes was more widespread than that for the hepatocytes grown on sandwich-cultured PS plates. The observation of ultrastructure in transmission electron microscopy clearly confirmed the presence of bile canalicular lumens possessing microvilli and tight junctions. Additionally, we demonstrated that the 7-ethoxyresorufin-O-deethylation activity of hepatocytes on PDMS membranes was also improved as compared to those on a PS surface. Therefore, sandwich-cultured hepatocytes on oxygen-permeable substrates can provide a simple tool for predicting the hepatic metabolism and toxicity of xenobiotics in vivo with short span and low cost in the course of drug discovery and evaluation.

Effectively organizing isolated cells to tissue elements having an appropriate microstructure is a fundamental issue in future tissue engineering, but biological cell-to-cell adhesion is too weak to assemble single cells directly. In order to overcome the difficulty, we applied an Avidin-Biotin Binding System (ABBS) to cell surfaces, and avidinylated and biotinylated cells could mutually bind in the short time they were mixed together. Unlike conventional intact cells, ABBS helped make larger spheroids. Interestingly, avidinylated and biotinylated cell adherence occurred within 1 sec using laser trapping, enabling single cell manipulation. We showed precise, direct single-cell-based tissue assembly using ABBS and optical tweezers, followed by damage-free tissue culture. The combination of ABBS and single cell manipulation has considerable potential for use in application such as tissue engineering, regenerative medicine, and drug screening system.

Using traditional 2-D photolithographic methods, surface patterns are made on agarose and used to form lipid vesicles with controlled size and layout. Depending on the size and layout of the patterned structures, the lipid bilayer vesicle size can be tuned and placement can be predetermined. Vesicles formed on 2-D patterned surfaces can be harvested for further investigations or can be assayed directly on the patterned surface. Lipid vesicles on the patterned surface are assayed for unilamellarity and protein incorporation, and vesicles are indeed unilamellar as observed from outer leaflet fluorescence quenching. Vesicles successfully incorporate the integral membrane protein α-hemolysin and maintain its membrane transport function.