研究者詳細 - 尾上　弘晃

論文 - 尾上　弘晃

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Adeno-Associated Virus-Encapsulated Alginate Microspheres Loaded in Collagen Gel Carriers for Localized Gene Transfer

Kurashina Y., Kurihara S., Kubota T., Takatsuka S., Hirabayashi M., Shimmura H., Miyahara H., Hioki A., Matsushita Y., Muramatsu J., Ogawa Y., Fujioka M., Okano H.J., Onoe H.

Advanced Healthcare Materials （Advanced Healthcare Materials） 13 （ 12 ） 2024年05月

ISSN 21922640

This work reports localized in vivo gene transfer by biodegradation of the adeno-associated virus-encapsulating alginate microspheres (AAV-AMs) loaded in collagen gel carriers. AAV-AMs are centrifugally synthesized by ejecting a mixed pre-gel solution of alginate and AAV to CaCl2 solution to form an ionically cross-linked hydrogel microsphere immediately. The AAV-AMs are able to preserve the AAV without diffusing out even after spreading them on the cells, and the AAV is released and transfected by the degradation of the alginate microsphere. In addition, AAV-AMs can be stored by cryopreservation until use. By implanting this highly convenient AAV-encapsulated hydrogel, AAV-AMs can be loaded into collagen gel carriers to fix the position of the implanted AAV-AMs and achieve localized gene transfer in vivo. In vivo experiments show that the AAV-AMs loaded in collagen gel carriers are demonstrated to release the encapsulated AAV for gene transfer in the buttocks muscles of mice. While conventional injections caused gene transfer to the entire surrounding tissue, the biodegradation of AAV-AMs shows that gene transfer is achieved locally to the muscles. This means that the proposed AAV-loaded system is shown to be a superior method for selective gene transfer.

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Light-Triggered Transmittance Control in Thermoresponsive Hydrogels by Femtosecond Laser Direct Writing

Kashikawa K., Tomikawa H., Onoe H., Terakawa M.

ACS Applied Optical Materials （ACS Applied Optical Materials） 2 （ 4 ） 565 - 573 2024年04月

Thermoresponsive hydrogels, which exhibit changes in their optical properties and volume due to temperature variations, are promising candidates for applications in soft devices. In this study, we demonstrate the modulation of transmittance in a thermoresponsive hydrogel through light stimulation employing gold microstructures fabricated via multiphoton photoreduction. The spatial integration of photoresponsiveness, attributed to high-density gold nanoparticles within the thermoresponsive hydrogel, was accomplished through the high-speed laser scanning of femtosecond laser pulses. The temperature measurement during the fabrication of the gold microstructure revealed that the high-speed and multiple scanning over the same path effectively reduced the temperature in the irradiated area of femtosecond laser pulses. The present approach enabled the mitigation of thermal effects during the fabrication, resulting in minimizing distortion in the fine lines of the structures. Upon exposure to stimulus light, a rapid change in the transmittance of the region where the structures were fabricated was prominently observed. The present method unveils a promising avenue for the advancement of light-responsive soft devices.

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In-line micro-spectrometer with a structural color polymer filter for the color and concentration monitoring system

Nishita S., Onoe H.

Sensors and Actuators A: Physical （Sensors and Actuators A: Physical） 367 2024年03月

ISSN 09244247

Monitoring systems for color and concentration of liquids have an important role in improving the fabrication process stably, not only in pipelines but also in microchannels. Our suggested in-line micro-spectrometer with an elastic structural color filter has detectability for color and concentration changes with a 40-mm size. The sequence of our device is the following: (1) the elastic structural color filter splits the white light from an LED, (2) the split light transmits the sample liquid, and (3) a photodiode receives the transmitted light. The structural color polymer is mechanically compressed inside the micro-spectrometer, leading to fixing the optical path even though the light wavelength is changed continuously. We fabricated the elastic structural color polymer with 200 nm silica nanoparticles and PEGPEA (poly- (ethylene glycol) phenyl ether acrylate). The prototype of the in-line micro-spectrometer with a red (λ = 596 nm) filter showed the detectability of the difference between yellow and green dyes. Moreover, the micro-spectrometer successfully distinguished the concentration of green-dyed water. This proposed in-line micro-spectrometer would contribute to developing in-line microfluidic color monitoring systems for biochemical analyses and material production.

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Mucin-Layer-Secreting in Vitro Intestinal Tube-Shaped Device with Cryptic Structure for Bacterial Co-Culture

Uramoto S., Tanaka S., Itai S., Onoe H.

Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems (MEMS) （Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems (MEMS)） 485 - 488 2024年

ISSN 10846999

We propose an in vitro intestinal tube-shaped device with a secreted mucin layer that enables us to achieve stable and continuous co-culturing with bacteria. Our tube-shaped device is made of collagen hydrogel and has 3D crypt-like structures created by electrolytically generated microbubbles. Bacterial suspension can be perfused via glass/silicone tubes attached at both ends of the tube device. Supply of culture medium diffused from the outside of the tube ensures a stable culture of Caco-2 and HT29 cells on the inside of the tube. We confirmed that HT29 cells mainly secreted a mucin layer in the apical portion of cryptic structures that spatially separates the intestinal cells and bacteria as seen in vivo environments. As a result, the co-culture of intestinal cells and lactobacillus in vitro was demonstrated in the 3D crypt-like structures in our intestinal tube-shaped device.

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Entirely Biodegradable Wireless pH Sensor with Split-Ring Resonators for Soil pH Monitoring

Sakabe K., Kan T., Onoe H.

Advanced Materials Technologies （Advanced Materials Technologies） 2024年

This paper describes a wireless and battery-free soil pH sensor made entirely of degradable materials. Soil pH monitoring is significant for precision agriculture and environmental conservation because soil health can be estimated by measuring soil pH. Soil pH can be measured by collecting soil samples and analyzing them in a laboratory environment or by inserting an electrode-type sensor into the soil and measuring soil pH on site. However, neither method is suitable for wide-area soil pH sensing for precision agriculture or environmental conservation. Herein, a fully biodegradable wireless pH sensor is presented that can be dispersed to outdoor environment to detect soil acidity. The sensor is operated wirelessly through split-ring resonators (SRRs) that consist of patterned octacalcium phosphate (OCP)-coated magnesium (Mg) on a poly-lactic acid (PLA) sheet. At acidic soil pH, the degradation of each SRR results in a change in the electromagnetic response of the sensor. The operation of the sensor in six different types of acidic soils is demonstrated to distinguish the acidic soil wirelessly. It is expected that the biodegradable sensor can be applied to low-cost, easy-to-use, and environmentally friendly monitoring of soil pH.

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Electromagnetic Metamaterial Absorbers for Soil Property Evaluation

Sato S., Sakabe K., Onoe H., Kan T.

ISSN 10846999

In this paper, we proposed a sensor probe utilizing an electromagnetic metamaterial absorber. This device comprises a tri-layer structure with periodically arranged copper split-ring resonators, a resin layer, and a magnesium layer, forming a complete electromagnetic wave absorber. Magnesium in contact with the soil dissolves in response to soil properties, and wireless measurement of the reflected response due to dissolution enables measurement of soil properties. Simulation and reflection measurement results revealed that this device can absorb 70% of incident electromagnetic waves. Furthermore, we examined the relationship between the reflection spectrum and the polarization direction, as well as the correlation between the time of device placement on soil and the reflection spectrum. The results showed that the device exhibited a low dependency on polarization direction and underwent significant changes in the reflection spectrum with the dissolution of magnesium. Therefore, this device shows a promising potential for a practical application in soil property evaluation.

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Dual-Sensing Mechanical Hydrogel Biosensor Composed by Aptamer Recognition and DNA Logic Gates

Kato S., Takinoue M., Onoe H.

ISSN 10846999

This paper presents a hydrogel biosensor that can simultaneously sense the co-existence of two substances through mechanical response using aptamer molecular recognition and DNA logic gates. Mechanical response is a preferable property in hydrogel sensors, so we have created a swelling hydrogel biosensor and have also added logical processing abilities to widen the applications of the sensor. We demonstrated the biosensor's ability against two targets, caffeine and theophylline, by measuring the swelling ratio of the hydrogel. We believe that our proposed hydrogel biosensor can be applied to many MEMS and μTAS applications because of its mechanical responsiveness and programmable reactions.

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Collagen Hollow Microbeads for Engineered Hollow Organ Model in Vitro

Abeta S., Masuda A., Hioki A., Shoji K., Onoe H.

ISSN 10846999

We propose an in vitro model to mimic the hollow structure of biological tissues (e.g. heart or stomach) using hollow core-shell collagen microgel beads. By using collagen hydrogel as the shell of the microbeads, cells encapsulated inside of the collagen shell can proliferate and form a tissue structure with a hollow center. In this paper, as a first step toward engineered hollow structure tissue formation, we report a fabrication method for core-shell (hollow) alginate microgel beads by a simple droplet-dipping method using a two-layer coaxial nozzle device. To apply collagen hydrogel to this droplet-dipping method, we applied alginate cross-linking network as a template for creating collagen microgel beads and dissolved the alginate network after the collagen hydrogel was stabilized. By combining these techniques, we believe that hollow collagen microgel beads encapsulating cells can easily and massively be produced for reconstructing in vitro hollow organ models.

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Macroscale Collagen-Actomyosin Hybrid Actuator Built from Bioderived Materials

Yoshida K., Kohno K., Hiratsuka Y., Onoe H.

Advanced Functional Materials （Advanced Functional Materials） 33 （ 51 ） 2023年12月

ISSN 1616301X

Biological muscles mainly consist of motor proteins that play essential roles and have a hierarchical structure covered with collagen that can transmit the tension to bones via tendons. The artificial reconstruction of motor proteins is highly expected from the viewpoint of engineering and biology. Although artificial muscles have been developed via synthetic and biological molecular motors, there is a considerable gap between the components of artificial and biological muscles, resulting in a significant obstacle to further scale-up and force transfer through macroscale structures. To overcome these obstacles via bioderived biomolecules, this study proposes a collagen-actomyosin hybrid soft actuator inspired by the structure of biological muscles. Collagen gel entrapping actomyosin can play roles in maintaining the actuator's shape and transmitting the tensile force. The generated forces reach the micro-newton range, enabling the actuation of millimeter-scale mechanical components. These properties may be helpful for the fabrication of soft robotic systems with advanced functionalities.

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Hydrogel-Sheathed hiPSC-Derived Heart Microtissue Enables Anchor-Free Contractile Force Measurement

Kurashina Y., Fukada K., Itai S., Akizuki S., Sato R., Masuda A., Tani H., Fujita J., Fukuda K., Tohyama S., Onoe H.

Advanced Science （Advanced Science） 10 （ 35 ） 2023年12月

In vitro reconstruction of highly mature engineered heart tissues (EHTs) is attempted for the selection of cardiotoxic drugs suitable for individual patients before administration. Mechanical contractile force generated in the EHTs is known to be a critical indicator for evaluating the EHT response. However, measuring contractile force requires anchoring the EHT in a tailored force-sensing cell culture chamber, causing technical difficulties in the stable evaluation of contractile force in long-term culture. This paper proposes a hydrogel-sheathed human induced pluripotent stem cell (hiPSC)-derived heart microtissue (H3M) that can provide an anchor-free contractile force measurement platform in commonly used multi-well plates. The contractile force associated with tissue formation and drug response is calculated by motion tracking and finite element analysis on the bending angle of the hydrogel sheath. From the experiment of the drug response, H3M is an excellent drug screening platform with high sensitivity and early testing capability compared to conventionally anchored EHT. This unique platform would be useful and versatile for regenerative therapy and drug discovery research in EHT.

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Schwann cell-encapsulated chitosan-collagen hydrogel nerve conduit promotes peripheral nerve regeneration in rodent sciatic nerve defect models

Takeya H., Itai S., Kimura H., Kurashina Y., Amemiya T., Nagoshi N., Iwamoto T., Sato K., Shibata S., Matsumoto M., Onoe H., Nakamura M.

Scientific Reports （Scientific Reports） 13 （ 1 ） 2023年12月

Chitosan has various tissue regeneration effects. This study was designed to investigate the nerve regeneration effect of Schwann cell (SC)-encapsulated chitosan-collagen hydrogel nerve conduit (CCN) transplanted into a rat model of sciatic nerve defect. We prepared a CCN consisting of an outer layer of chitosan hydrogel and an inner layer of collagen hydrogel to encapsulate the intended cells. Rats with a 10-mm sciatic nerve defect were treated with SCs encapsulated in CCN (CCN+), CCN without SCs (CCN−), SC-encapsulated silicone tube (silicone+), and autologous nerve transplanting (auto). Behavioral and histological analyses indicated that motor functional recovery, axonal regrowth, and myelination of the CCN+ group were superior to those of the CCN− and silicone+ groups. Meanwhile, the CCN− and silicone+ groups showed no significant differences in the recovery of motor function and nerve histological restoration. In conclusion, SC-encapsulated CCN has a synergistic effect on peripheral nerve regeneration, especially axonal regrowth and remyelination of host SCs. In the early phase after transplantation, SC-encapsulated CCNs have a positive effect on recovery. Therefore, using SC-encapsulated CCNs may be a promising approach for massive peripheral nerve defects.

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Spatiotemporal single-cell tracking analysis in 3D tissues to reveal heterogeneous cellular response to mechanical stimuli

Kasahara K., Muramatsu J., Kurashina Y., Miura S., Miyata S., Onoe H.

Science Advances （Science Advances） 9 （ 41 ） 2023年10月

Mechanical stimuli have been recognized as important for tissue maturation, homeostasis and constructing engineered three-dimensional (3D) tissues. However, we know little about the cellular mechanical response in tissues that could be considerably heterogeneous and spatiotemporally dynamic due to the complex structure of tissues. Here, we report a spatiotemporal single-cell tracking analysis of in vitro 3D tissues under mechanical stretch, to reveal the heterogeneous cellular behavior by using a developed stretch and optical live imaging system. The system could affect the cellular orientation and directly measure the distance of cells in in vitro 3D myoblast tissues (3DMTs) at the single-cell level. Moreover, we observed the spatiotemporal heterogeneous cellular locomotion and shape changes under mechanical stretch in 3DMTs. This single-cell tracking analysis can become a principal method to investigate the heterogeneous cellular response in tissues and provide insights that conventional analyses have not yet offered.

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Spatially-selective enhancement of mechanical strength through localized cross-linking of double network hydrogels

Tsunemitsu K., Watanabe A., Onoe H., Terakawa M.

Optical Materials Express （Optical Materials Express） 13 （ 10 ） 2914 - 2924 2023年10月

To advance the development of flexible materials for soft robotics applications, it is crucial to enhance the elastic modulus and breaking the stress of soft materials, such as hydrogels. Double network hydrogels (DN gels) have displayed remarkable mechanical strength owing to their unique network structure composed of two types of polymer networks. However, current fabrication methods for DN gels entail cross-linking two distinct hydrogel polymers throughout the entire hydrogel matrix. In this study, we focused on employing multi-photon polymerization (MPP) with femtosecond laser pulses as a cross-linking method for hydrogels to achieve spatially selective formation of DN gel structures at the micrometer scale, along with the consequent improvement in local mechanical strength. We assessed the mechanical properties of the fabricated structures and confirmed that the mechanical strength varied within the regions where DN gel was locally formed.

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Unusual Selective Monitoring of N,N-Dimethylformamide in a Two-Dimensional Material Field-Effect Transistor

Fukui A., Matsuyama K., Onoe H., Itai S., Ikeno H., Hiraoka S., Hiura K., Hijikata Y., Pirillo J., Nagata T., Takei K., Yoshimura T., Fujimura N., Kiriya D.

ACS Nano （ACS Nano） 17 （ 15 ） 14981 - 14989 2023年08月

ISSN 19360851

N,N-Dimethylformamide (DMF) is an essential solvent in industries and pharmaceutics. Its market size range was estimated to be 2 billion U.S. dollars in 2022. Monitoring DMF in solution environments in real time is significant because of its toxicity. However, DMF is not a redox-active molecule; therefore, selective monitoring of DMF in solutions, especially in polar aqueous solutions, in real time is extremely difficult. In this paper, we propose a selective DMF sensor using a molybdenum disulfide (MoS2) field-effect transistor (FET). The sensor responds to DMF molecules but not to similar molecules of formamide, N,N-diethylformamide, and N,N-dimethylacetamide. The plausible atomic mechanism is the oxygen substitution sites on MoS2, on which the DMF molecule shows an exceptional orientation. The thin structure of MoS2-FET can be incorporated into a microfluidic chamber, which leads to DMF monitoring in real time by exchanging solutions subsequently. The designed device shows DMF monitoring in NaCl ionic solutions from 1 to 200 μL/mL. This work proposes the concept of selectively monitoring redox-inactive molecules based on the nonideal atomic affinity site on the surface of two-dimensional semiconductors.

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Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Takatsuka S., Kubota T., Kurashina Y., Kurihara S., Hirabayashi M., Fujioka M., Okano H.J., Onoe H.

Biotechnology and Bioengineering （Biotechnology and Bioengineering） 120 （ 8 ） 2371 - 2377 2023年08月

ISSN 00063592

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l-lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.

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L-2L ladder digital-to-analogue converter for dynamics generation of chemical concentrations

Chatani T., Shiraishi S., Miyazako H., Onoe H., Hori Y.

Royal Society Open Science （Royal Society Open Science） 10 （ 4 ） 2023年04月

Cellular response to dynamic chemical stimulation encodes rich information about the underlying reaction pathways and their kinetics. Microfluidic chemical stimulators play a key role in generating dynamic concentration waveforms by mixing several aqueous solutions. In this article, we propose a multi-layer microfluidic chemical stimulator capable of modulating chemical concentrations by a simple binary logic based on the electronic-hydraulic analogy of electronic R-2R ladder circuits. The proposed device, which we call L-2L ladder digital-to-analogue converter (DAC), allows us to systematically modulate 2n levels of concentrations from single sources of solution and solvent by a single operation of 2n membrane valves, which contrasts with existing devices that require complex channel geometry with multiple input sources and valve operations. We fabricated the L-2L ladder DAC with n = 3 bit resolution and verified the concept by comparing the generated waveforms with computational simulations. The response time of the proposed DAC was within the order of seconds because of its simple operation logic of membrane valves. Furthermore, detailed analysis of the waveforms revealed that the transient concentration can be systematically predicted by a simple addition of the transient waveforms of 2n = 6 base patterns, enabling facile optimization of the channel geometry to fine-tune the output waveforms.

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Near-Infrared-Triggered On-Demand Controlled Release of Adeno-Associated Virus from Alginate Hydrogel Microbeads with Heat Transducer for Gene Therapy

Takatsuka S., Kubota T., Kurashina Y., Onoe H.

Small （Small） 19 （ 7 ） 2023年02月

ISSN 16136810

Gene therapy using adeno-associated virus (AAV) has potential as a radical treatment modality for genetic diseases such as sensorineural deafness. To establish clinical applications, it is necessary to avoid immune response to AAV by controlled release system of AAV. Here, a near-infrared (NIR)-triggered on-demand AAV release system using alginate hydrogel microbeads with a heat transducer is proposed. By using a centrifuge-based microdroplet shooting device, the microbeads encapsulating AAV with Fe3O4 microparticles (Fe3O4-MPs) as a heat transducer are fabricated. Fe3O4-MPs generated heat by NIR enhanced the diffusion speed of the AAV, resulting in the AAV being released from the microbeads. By irradiating the microbeads encapsulating fluorescent polystyrene nanoparticles (FP-NPs) (viral model) with NIR, the fluorescence intensity decreased only for FP-NPs with a diameter of 20 nm and not for 100 or 200 nm, confirming that this system can release virus with a diameter of several tens of nanometers. By irradiating NIR to the AAV-encapsulating microbeads with Fe3O4-MPs, the AAV is released on demand, and gene transfection to cells by AAV is confirmed without loss of viral activity. The NIR-triggered AAV release system proposed in this study increases the number of alternatives for the method of drug release in gene therapy.

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A Novel Rat Model of Embolic Cerebral Ischemia Using a Cell-Implantable Radiopaque Hydrogel Microfiber

Komatsu T., Ohta H., Takakura N., Hata J., Kitagawa T., Kurashina Y., Onoe H., Okano H.J., Iguchi Y.

Translational Stroke Research （Translational Stroke Research） 2023年

ISSN 18684483

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.

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Oil-Sealed Rgd-Modified Hydrogel Microwell Array with Size-Selective Permeation for Analysis on Exosomes from Single Cells

Yamagata C., Itai S., Kurashina Y., Asai M., Hoshino A., Onoe H.

ISSN 10846999

We propose an oil-sealed arginine-glycine-aspartate (RGD)-modified hydrogel microwell array for analyzing exosomes secreted from single cells. Our device realizes the collection of exosomes from single cells in parallel. The size-selective permeation of the RGD-modified alginate hydrogel allows both stable cell culturing of single or few cells in closed wells and the confinement of exosomes secreted in each space. The nutrients (< 20 nm) that are necessary for cell culture can pass through the hydrogel, while exosomes (30-150 nm) do not permeate the walls of microwells. The cell culture property and the permeability of our device were examined, showing the capability to collect exosomes from single cells. We believe that our device would contribute to understanding the mechanisms of various diseases.

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In-Ice Polymerization for Functional Hydrogel Microbead with Flash Freezing Centrifugal Microfluidic Device

Murayama T., Yoshida K., Kurashina Y., Onoe H.

ISSN 10846999

This paper describes a centrifuge-based fabrication method of microgel beads using flash freezing in liquid nitrogen and in-ice polymerization. Previously, materials for fabricating microgel beads with centrifugal microfluidic systems have been limited to only sodium alginate solution using rapid ionic cross-linking. By using our proposed method, the shape of beads can be defined by flash freezing of the ejected pre-gel microdroplets in liquid nitrogen and successive in-ice polymerization by UV irradiation. We succeeded in fabricating ethanol-responsive structural-color beads and confirmed their ethanol responsivity by exhibiting visible structural color change. Our fabrication method will expand the variety of materials for the centrifugal microfluidic microgel beads fabrication method and possible applications including biosensors and drug delivery systems.

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尾上 弘晃

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尾上　弘晃

論文 - 尾上　弘晃