Published papers

We proposed an oil-sealed hydrogel microwell array for analysis of secretory components from isolated single cells. The purpose of our device is to realize the isolation of the secretory components during cell culture by combining the semi-permeable arginine-glycine-aspartate (RGD)-modified calcium alginate microwell and the oil sealing. We demonstrated the oil-sealing property, the impermeability to extracellular vesicles (EVs) or growth factor proteins (GFs), and the permeability to nutrients of our device by using three types of fluorescent particles with different diameters. We believe that our oil-sealed hydrogel microwell array will contribute to biomedical analysis on single-cell metabolism that helps to understand the mechanisms of various diseases.

This paper presents a fully biodegradable wireless sensor for measurements of soil urea over a large area. The sensor consists of an alginate hydrogel with immobilized urease enzyme and a metamaterial perfect absorber. Our sensor has the capability to detect the urea concentration in soil wirelessly. In this paper, we succeeded in wirelessly and robustly detecting the soil urea by measuring time-transient changes of electromagnetic responses from the biodegradable soil urea sensors placed on the urea-containing soils. This proposed sensor would enable continuous but also robust measurements for soil urea monitoring over a large area.

This paper describes a fabrication method of an in vitro branched vascular model using extracellular matrix (ECM)-derived materials (transglutaminase crosslinked gelatin, TG-gelatin). Mechanical stresses, such as disturbed blood flow derived from vascular branches, are a significant cause of cardiovascular disease. To study cardiovascular diseases, a perfusion and stretching culture platform with an ECM-based in vitro vascular model with branches has been essential. Among the proposed microchannel fabrication methods, sacrificial molding with a template made of soluble material is attractive for fabricating branched microchannels. However, the soluble template remained an issue of undesired deformation due to the swelling caused by the moisture in the hydrogel. Here, we propose groove-aided sacrificial molding (GAS molding) to suppress the deformation of the soluble template in the TG-gelatin. By preparing a channel-shaped groove on the TG-gelatin, the shape retention of the soluble template is assisted. We experimentally evaluated the deformation of the microchannels fabricated by the proposed GAS molding. The vascular endothelial cells were seeded into microchannels fabricated by GAS molding for perfusion and stretching culture. The effects of mechanical stress were visualized by immunofluorescence staining of PECAM1 and integrin α9 in the endothelial cells. Overall, our method would suggest a platform to spatially elucidate the cellular responses to irregular mechanical stresses, such as triggers of cardiovascular disease.

This paper describes efficient light-driven self-assembly by high-SA/V (Surface Area to Volume)-ratio TiO2-nanoparitcle-encapsulating hydrogel unit. The shape of the hydrogel units can be designed to achieve complex and efficient self-assembly system. Our TiO2-nanoparticle-encapsulating hydrogel unit has a high SA/V ratio rather than TiO2 particles, bringing a high ion concentration gradient and large driving force. We achieved the velocity control of silica colloids by changing TiO2-nanoparticle-concentration and demonstrated a silica colloid accumulator with our hydrogel unit. We believe that our efficient light-driven self-assembly could applied to a micromotor with complex motion and a functionality microdevice for using in nature.

Detection of chemical substances is essential for living a healthy and cultural life in the modern world. One type of chemical sensing technology, biosensing, uses biological components with molecular recognition abilities, enabling a broad spectrum of sensing targets. Short single-stranded nucleic acids called aptamers are one of the biological molecules used in biosensing, and sensing methods combining aptamers and hydrogels have been researched for simple sensing applications. In this research, we propose a hydrogel-based biosensor that uses aptamer recognition and DNA-driven swelling hydrogels for the rapid detection of histamine. Aptamer recognition and DNA-driven swelling hydrogels are directly linked via DNA molecular reactions, enabling rapid sensing. We selected histamine, a major food poisoning toxin, as our sensing target and detected the existence of histamine within 10 min with significance. Because this sensing foundation uses aptamers, which have a vast library of targets, we believe this system can be expanded to various targets, broadening the application of hydrogel-based biosensors.

Hydrogel-based drug carriers provide on-demand drug release via external stimuli. Ultrasound is a promising method because of the potential for remotely releasing the drug. However, intense ultrasound irradiation has been required in previous studies. This paper reports drug model release from hydrogel carriers encapsulating bubble vibration enhancers (BVEs) consisting of microbubbles coated with a lipid membrane. Vibration of BVEs induced by ultrasound stimulation promoted the release of drug models with ultrasound irradiation controlled to a biologically safe acoustic pressure based on spatial-peak temporal-average intensity (ISPTA). The release ratio increased significantly from 2.3 % without BVEs and ultrasound to 10.2 % with both. To evaluate the frequency response, the release ratio was measured at three different ultrasound frequencies (0.3, 1.8, and 2.5 MHz), showing increased efficiency as the frequency approached the resonance frequency of the BVEs. For in vivo applications, hydrogel microspherical carriers with BVEs achieved a 12 % release ratio. Poly-L-lysine coating successfully suppressed the drug release to 0.2 %. The carriers demonstrated repeated responsiveness when ultrasound was applied in three 5-minute intervals. The hydrogel carrier encapsulating BVEs we proposed is a promising in vivo device capable of releasing drugs on demand by ultrasound irradiation based on its high biosafety and acoustic responsiveness.

This paper describes a hydrogel microneedle patch with arrayed colorimetric sensing microbeads that detect the glucose concentration of bodily fluids. The PEGDA hydrogel microneedle swallows bodily fluid from the stratum corneum inducing a reaction from the glucose sensing TiO2 calcium alginate microbeads, encapsulating enzymes and dyes; showing a clear color change in accordance with the spatial glucose concentration of the absorbed fluid. We have examined the sensing characteristics of our microneedle patch and its applicability to transdermal sensing by using a hydrogel skin model containing glucose fluid. We believe that this microneedle allows for a faster, more efficient, and easier sensing as a bodily sensor.

This paper reports a hydrogel microwell array device capable of long-term (over 144 hours) gas-phase culture of olfactory receptor expressing cells in an ambient environment while retaining odor responsiveness. By seeding cells onto a hydrogel that allows liquid permeability, the liquid level inside the wells lowers due to atmospheric pressure, while a thin film of medium is maintained by surface tension. This enables the maintenance of a liquid film thin enough to allow odor responsiveness while continuously supplying nutrients to the cells in an ambient environment. This device enables simultaneous cell culture and odor sensing in an ambient environment, making it applicable as an odorant sensor.

Poly(para-xylylene)s (PPXs), or so-called parylenes, have become a well-established polymer class in the conformal coating industry. Due to their transparent nature, low electrical conductivity, and high biocompatibility, they are ideal candidates for coating medical devices and other delicate electronics. However, the crosslinking of PPXs to enhance their durability is still challenging today. Expensive setups need to be used to obtain crosslinked PPXs. Furthermore, the possibility of functionalization post-polymerization is limited. In this work, we present the synthesis of a hydrosilane functionalized [2.2]paracyclophane, which is used to obtain the corresponding hydrosilane functionalized poly(para-xylylene) (PPX-SiH). Through the formation of siloxane bonds during the low-pressure chemical vapor deposition (LP-CVD), which increases internal bonding, PPX-SiH is obtained as a flexible and durable polymer film. The siloxane formation during the LP-CVD is investigated through X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance spectroscopy (NMR), and Fourier-transform infrared spectroscopy (FTIR). Additionally, mechanistic insights into the formation of siloxane bonds are given through quantum chemical calculation. The Si-H bonds in the polymer allow for oxidation to form bridging siloxane moieties which enhances stretchability while also increasing the resistance to organic solvents. Through the passivation of the surface during oxygen plasma treatment, PPX-SiH becomes practically plasma-etch-resistant.

This paper proposes a two-layered hollow hydrogel microfiber for cell therapy. Our proposed hydrogel microfiber has a tube-like hollow structure with embedded tissues, enabling to be retained in a blood vessel for a long period of time without interfering with blood flow. In this paper, as a proof of concept, we examined hollow hydrogel microfibers for intravascular delivery without blood flow interruption. By using a two-layered co-axial microfluidic device, a hollow hydrogel microfiber was formed. The hollow microfiber was delivered into a blood vessel model and shown to be fixed without interrupting fluid flow in the blood vessel model. Furthermore, as an in vivo test using a rat, the hollow fibers with X-ray visibility were delivered to rat kidneys with the angiographic agent, Lipiodol, to confirm that the hollow fibers could be retained in vivo without interfering with blood flow. We believe that our proposed hollow hydrogel microfiber would be an effective intravascular cell-delivery platform in clinical practice for cell therapy.

We report on an isotropic degradable metamaterial (IDM) that is a sensor probe for environmental monitoring, such as a wet soil condition. The isotropic degradable metamaterial was designed like a pebble and can be scattered on the soil. When the metal pattern on the isotropic degradable metamaterial is degraded by the chemical reaction between the metal and the surroundings, the isotropic degradable metamaterial resonant behavior vastly changes. Environmental information thus can be measured from the isotropic degradable metamaterial reflection wirelessly. Considering the sensing situation, we derived an X-shaped metal isotropic degradable metamaterial design that provides resonant reflection spectra unchanged regardless of the posture of the isotropic degradable metamaterial. Based on the simulation results, we fabricated the isotropic degradable metamaterial that operates in the 2.4 GHz band. The microwave measurements revealed that the isotropic degradable metamaterial presented an isotropic response; the reflectance spectra did not change by the postures. Furthermore, the reflectance spectra could read out the resonant metal (Mg) degradation with water, regarded as an environmental condition. These results indicate the potential application of the proposed isotropic degradable metamaterial as an environmental sensor probe.

Vasculogenic mimicry (VM) is a process by which tumor cells form vessel-like network to secure oxygen and nutrients essential for tumor growth. Intercellular junctions, including tight junctions, may play a critical role in this process, important for VM. Here, we investigated the role of angulin-1/LSR (Ang-1), a key component of tricellular tight junctions, in VM regulation. We performed VM assay using Ang-1 KO breast cancer cells. Compared with control cells, Ang-1/KO cells exhibited significantly enhanced VM formation, accompanied by increasing epithelial-mesenchymal transition features. In a xenograft model, tumors derived from Ang-1/KO cells showed increased mass and elevated VM ratios. Re-expression of Ang-1 isoforms 3, 4, or 6-2 in Ang-1/KO cells suppressed VM formation, whereas isoforms 1, 2, and 6 had no effect. RT-PCR analysis of breast cancer patient tissues confirmed the lower expression of Ang-1 isoform 6-2, which suppresses VM. These findings identify Ang-1 as a novel regulator of VM and suggest that specific isoforms, particularly isoform 6-2, may serve as isoform-specific diagnostic markers or therapeutic targets in breast cancer.

This paper proposes the controlled release of adeno-associated virus (AAV) by polymer mesh structures of alginate hydrogel microbeads (gel-beads). The polymer mesh structure of alginate hydrogel can be controlled by adjusting the number of cross-linking points depending on the molecular structure (M/G ratio) of the alginate hydrogel. Scanning electron microscopic analysis on the mesh structures of alginate hydrogels with M/G ratios of 0.96 (MG-0.96) and 0.42 (MG-0.42) indicates that the mesh size distribution of MG-0.42 hydrogel was smaller than that of MG-0.96 hydrogel. Using these characteristics, controlled release of AAVs (AAV-1 or AAV-5) from M/G-ratio-tuned gel-beads (MG-0.96 or MG-0.42), Janus-shaped gel-beads consisting of hemispheres of the MG-0.96 and MG-0.42, and MG-0.64 gel-beads consisting of a mixture of MG-0.96 and MG-0.42 were examined with ELISA for AAV capsids and the GFP fluorescent signals from the transfected cells. The precise control of AAV release by the mesh structure of the alginate hydrogels could be an effective method for creating AAV micro-carriers for gene therapy.

Here an electrical stimulation system is described for maturing microfiber-shaped cardiac tissue (cardiac microfibers, CMFs). The system enables stable culturing of CMFs with electrical stimulation by placing the tissue between electrodes. The electrical stimulation device provides an electric field covering whole CMFs within the stimulation area and can control the beating of the cardiac microfibers. In addition, CMFs under electrical stimulation with different frequencies are examined to evaluate the maturation levels by their sarcomere lengths, electrophysiological characteristics, and gene expression. Sarcomere elongation (14% increase compared to control) is observed at day 10, and a significant upregulation of electrodynamic properties such as gap junction protein alpha 1 (GJA1) and potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) (maximum fourfold increase compared to control) is observed at day 30. These results suggest that electrically stimulated cultures can accelerate the maturation of microfiber-shaped cardiac tissues compared to those without electrical stimulation. This model will contribute to the pathological research of unexplained cardiac diseases and pharmacologic testing by stably constructing matured CMFs.

This paper proposes a fibrin-based actomyosin soft actuator with excellent mechanical strength. The actuator is composed of motor proteins derived from chicken's skeletal muscle tissues and is driven by converting chemical energy from adenosine triphosphate (ATP) into kinetic energy. Furthermore, it is superior to the collagen-based actomyosin soft actuator in a previous study in terms of toughness and protection of actomyosin from thermal damage. We succeeded in fabricating the fibrin-based actomyosin soft actuator and their drive by ATP. The Young's modulus of the actuator was 15 kPa that is a significant improvement compared to a collagen actuator (<1 kPa). This study is expected to contribute to the realization of motor proteins bottom-up soft actuators that imitates the molecular structure and actuation of skeletal muscle.

Soft actuators have developed over the last decade for diverse applications including industrial machines and biomedical devices. Integration of chemical sensors with soft actuators would be beneficial in analyzing chemical and environmental conditions, but there have been limited devices to achieve such sensing capabilities. In this work, we developed a thin-film soft actuator integrated with a paper-based chemical sensor, termed a microfluidic paper-based analytical soft actuator (μPAC). μPAC consists of (1) a silicone thin film with a 3D-printed pneumatic chamber and (2) a cellulose paper. This cellulose paper offers dual functions: the strain-limiting layer of a soft actuator and the substrate for the chemical sensor for a paper-based analytical device (μPAD). We characterized the design parameters of the actuators—namely, (1) thickness of silicone thin film, (2) chamber length, and (3) Young's modulus of silicone thin film—to evaluate the actuation performance. These characterizations suggested that the cellulose paper served as a suitable self-straining layer of the actuator, making μPAC a chemical sensor that can actuate simultaneously. Highlighting the unique capability of μPAC, we demonstrated the local detection of pH on the curved target surface. Overall, this research demonstrated the rapid fabrication of actuating chemical sensors with a unique design by combining soft actuators and μPAD, enabling chemical sensing on various surface topologies by dynamically making conformal contact.

We propose an oil-sealed, arginine-glycine-aspartic (RGD) -modified alginate hydrogel microwell array for the analysis of single-cell-derived extracellular vesicles and particles (EVPs) secreted by adherent cells cultured in enclosed spaces. Taking advantage of the mesh structure of alginate hydrogel, we developed a microwell array with size-selective permeability that allows nutrients to pass through the hydrogel while preventing EVPs from doing so. Continuous single-cell culture in sealed microwells (>19 days) has been achieved, while retaining the EVPs inside the microwells. The single-cell-derived EVPs were collected from sealed microwells using a glass capillary to analyze surface membrane proteins. We believe that our oil-sealed RGD-modified alginate hydrogel microwell array will contribute to revealing the heterogeneity of cells, thereby advancing our understanding of the mechanisms of various diseases.

The failure of neuroprotective treatment-related clinical trials, including stem cell therapies, may be partially due to a lack of suitable animal models. We have developed a stem cell-implantable radiopaque hydrogel microfiber that can survive for a long time in vivo. The microfiber is made of barium alginate hydrogel containing zirconium dioxide, fabricated in a dual coaxial laminar flow microfluidic device. We aimed to develop a novel focal stroke model using this microfiber. Using male Sprague-Dawley rats (n=14), a catheter (inner diameter, 0.42 mm; outer diameter, 0.55 mm) was navigated from the caudal ventral artery to the left internal carotid artery using digital subtraction angiography. A radiopaque hydrogel microfiber (diameter, 0.4 mm; length, 1 mm) was advanced through the catheter by slow injection of heparinized physiological saline to establish local occlusion. Both 9.4-T magnetic resonance imaging at 3 and 6 h and 2% 2,3,5-triphenyl tetrazolium chloride staining at 24 h after stroke model creation were performed. Neurological deficit score and body temperature were measured. The anterior cerebral artery-middle cerebral artery bifurcation was selectively embolized in all rats. Median operating time was 4 min (interquartile range [IQR], 3–8 min). Mean infarct volume was 388 mm3 (IQR, 354–420 mm3) at 24 h after occlusion. No infarction of the thalamus or hypothalamus was seen. Body temperature did not change significantly over time (P = 0.204). However, neurological deficit scores before and at 3, 6, and 24 h after model creation differed significantly (P < 0.001). We present a novel rat model of focal infarct restricted to the middle cerebral artery territory using a radiopaque hydrogel microfiber positioned under fluoroscopic guidance. By comparing the use of stem cell-containing versus non-containing fibers in this stroke model, it would be possible to determine the efficacy of "pure" cell transplantation in treating stroke.

Disruption of the microbiome in humans caused several diseases. To infer causality, the intestinal models are essential, but the commonly used model has still room for improvements such as biomimicry, productivity, and ability to co-culture with bacteria. This paper describes the artificial intestinal tissue by culturing intestinal cells (Caco-2) in the collagen gel tube with crypt-like cavities created by electrolysis-triggered microbubbles. This tube device has high biomimicry and productivity and also consists of glass/silicone tubes connector at both ends, which allows for connecting external pumps easily and running two types of flows: the inside of the tube (internal flow) and permeation from the outside of the tube to the inside (external flow). In order to show this high extensibility, we demonstrated the co-culture of Caco-2 cells and bacteria (Bifidobacteria). We believe that our device will be a platform for investigating complex intestinal diseases due to its high biomimetic properties.

We investigated the relationship between the deformation of the molecular network of myosin and actin and the macroscopic actuator contraction. Although several bioactuators using actomyosin have been reported in the past, the relationship between the molecular-scale structure and the motion of actomyosin actuators remains largely unexplored. We stained for actin, myosin, and collagen, respectively, and observed the internal biomolecular structure of the actuator by fluorescence observation. We took time-lapse images of the contraction of fluorescently stained actuators and determined the molecular contraction ratio of actomyosin. Finally, the macro and micro contraction ratios of the actuators were compared and examined to understand the internal mechanism of the actomyosin-collagen bioactuator.