Details of a Researcher - Onoe, Hiroaki

Papers - Onoe, Hiroaki

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Shape Retaining and Sacrificial Molding Fabrication Method for ECM-Based in vitro Vascular Model

Muramatsu J., Goh W.H., Shimizu A., Hashimoto K., Hashimoto M., Miura 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)) 2021-January 230 - 233 2021.01

ISSN 9781665419123

This paper describes a new fabrication method for the extracellular matrix (ECM)-based in vitro vascular model. The vascular model was fabricated with shape retaining mold and sacrificial mold (shape retaining and sacrificial molding, SRS-molding). We compared SRS-molding and sacrificial molding. In addition, complex mechanical stimuli such as stretching and flowing in the branches of the vascular model were analyzed by simulation software. Finally, we performed the perfusion culturing with our vascular model and stretching them. Our vascular model and mechanical stimuli analysis would elevate the vascular culturing to the next step.

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Real-Time Three-Dimensional Single-Cell-Resolution Monitoring System for Observation of Dynamic Cell Behavior under Mechanical Stimuli

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

ISSN 9781665419123

We present an experimental system for real-time monitoring of 3D constructed tissues at single-cell resolution to further understand cellular response against mechanical stimuli. The system enables us to continuously monitor the same cells in a 3D tissue at a subcellular level under uniaxial cyclic stretch. We confirmed our system's effectiveness by observing that 3D tissues cultured under cyclic stretch were oriented to the direction of stretch. Moreover, cells in a 3D tissue were successfully observed continuously at single-cell resolution even under cyclic stretch. We believe that the real-time observation of cellular response against mechanical stimuli with our demonstrated system could be an effective method for investigating the relationship between cellular response and mechanical stimuli in detail.

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On-The-Fly Rapid-Cooling Gelation of Microgel Beads in Centrifugal Microfluidic Device with Liquid Nitrogen

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

ISSN 9781665419123

This paper describes a new fabrication method of microgel beads crosslinked by rapid temperature change using a centrifugal microfluidic device with a heating system and liquid nitrogen. Previously, materials for fabricating microgel beads with centrifugal microfluidic systems is limited to only sodium alginate solution. By using our proposed method, microgel beads could be fabricated by rapidly cooling pre-gel droplet ejected by centrifugal force. We succeeded in fabricating agarose microgel beads by rapid gelation in the liquid nitrogen. Our fabrication method will expand the variety of materials for the centrifugal microfluidic microgel beads fabrication method and possible applications including biosensors, tissue engineering and drug delivery systems.

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Ethanol Driven Micro-Robots with Photonic Colloidal Crystal Hydrogel for Exploring and Sensing Stimuli

Yoshida K., Yamawaki S., Onoe H.

ISSN 9781665419123

Photonic colloidal crystal hydrogels are expected to be a powerful tool for environmental monitoring and healthcare. The photonic colloidal crystal hydrogel reflects visible-light, so the intensity of the stimulus can be converted to visible color. However, these sensors required taking a measured sample from the target environment. To detect a specific substance in a vast environment, exploring and detecting systems are required. Therefore, we proposed ethanol driven micro-robots with photonic colloidal crystal hydrogel. We achieved the color changes of micro-robots response to the external stimuli. Proposed micro-robots propelled over 600 s by the Marangoni effect caused by ethanol diffusion.

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Electrical Field Chamber with Dinamically Adaptable Stimuli for Myocyte Orientation during Cultivation

Ramirez S.R.M., Yamada T., Funahashi A., Onoe H.

ISSN 9781665419123

Tissue engineering is a promising technology for both its possible applications in the fields of regenerative medicine and medical research. One of its challenges is to create an environment which allows the cell cultures to develop correctly. This environment is far from static and normally is modified by the cells themselves. To this end, adaptable systems have to be developed to ensure a successful development of the in-vitro tissue. This research focuses on a system which is able to modify an external electrical stimulus on a culture chamber, during its cultivation. This system, through a variation of the stimulus direction, successfully improved the orientation of the developing myocytes.

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Anionic fluorophore-assisted fabrication of gold microstructures inside a hydrogel by multi-photon photoreduction

MACHIDA M., SHEN W., ONOE H., HIRUTA Y.U.K.I., HEISTERKAMP A., MAZUR E.R.I.C., TERAKAWA M.

Optical Materials Express (Optical Materials Express) 11 ( 1 ) 48 - 58 2021.01

The fabrication of accentuated gold microstructures is demonstrated by multi-photon photoreduction inside an anionic fluorophore-containing hydrogel. We attempted to facilitate gold-ion photoreduction near the focal point of laser pulses, expecting the donation of electrons by the oxidation of fluorophores in the vicinity of gold ions. The presence of anionic FITCdextran also inhibited the spontaneous reduction in untargeted zones, which is attributed to the coordination of gold ions and the anionic FITC-dextran. Simultaneous facilitation and inhibition are promising for the fabrication of dense metal microstructures in the targeted zone while maintaining the hydrogel's light permeability.

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Travelling ultrasound promotes vasculogenesis of three-dimensional-monocultured human umbilical vein endothelial cells

Imashiro C., Azuma T., Itai S., Kuribara T., Totani K., Onoe H., Takemura K.

Biotechnology and Bioengineering (Biotechnology and Bioengineering) 118 ( 10 ) 3760 - 3769 2021

ISSN 00063592

To generate three-dimensional tissue in vitro, promoting vasculogenesis in cell aggregates is an important factor. Here, we found that ultrasound promoted vasculogenesis of human umbilical vein endothelial cells (HUVECs). Promotion of HUVEC network formation and lumen formation were observed using our method. In addition to morphological evaluations, protein expression was quantified by western blot assays. As a result, expression of proteins related to vasculogenesis and the response to mechanical stress on cells was enhanced by exposure to ultrasound. Although several previous studies have shown that ultrasound may promote vasculogenesis, the effect of ultrasound was unclear because of unregulated ultrasound, the complex culture environment, or two-dimensional-cultured HUVECs that cannot form a lumen structure. In this study, regulated ultrasound was propagated on three-dimensional-monocultured HUVECs, which clarified the effect of ultrasound on vasculogenesis. We believe this finding may be an innovation in the tissue engineering field.

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Charged fluorophores-assisted fabrication of metallic structures inside hydrogel by multi-photon photoreduction

MacHida M., Shen W., Onoe H., Heisterkamp A., Mazur E., Terakawa M.

Proceedings of SPIE - The International Society for Optical Engineering (Proceedings of SPIE - The International Society for Optical Engineering) 11676 2021

ISSN 9781510641877

We demonstrated the fabrication of gold microstructures inside a PEG-based hydrogel with different charged fluorophores by multi-photon photoreduction. By adding an anionic charged FITC-dextran or a cationic charged Rhodamine110 to the metal ion solution, line width of the fabricated gold microstructures increased compared to that of the structures fabricated in fluorophore-free hydrogel. The photobleaching could enhanced the reduction of gold ions accompanying the oxidation of the fluorophores. Notably, the inhibition of reduction of gold ions at a site other than the focal point was observed with FITC-dextran, which may be attributable to the coordination of gold ions to FITC-dextran.

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ATP-DRIVEN MUSCLE INTEGRATED MICROROBOT ACTUATED BY TRACTION OF ACTOMYOSIN-COLLAGEN HIBRID HYDROGEL

Kohno K., Hiratsuka Y., Onoe H.

MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences) 619 - 620 2021

We propose an elastomeric microrobot actuated by ATP-driven biological traction of actomyosin. Our robot is composed of a PDMS thin film part as a body and an actomyosin-embedded collagen hydrogel part as a linear motor. The actomyosin drives the hydrogel linear motor by the hydrolysis of ATP. This actomyosin linear motor could repeatably contract by adding ATP and stretching similar to the driving mechanism of our body. By combining the linear motor part and PDMS actuator part, we succeeded in driving the whole device by the traction force of actomyosin. The traction force generated by the linear motor was estimated from the deformation of the PDMS body. We believe that our device would be a new approach to apply biological motor for micro actuators.

ULTRASOUND-TRIGGERED ON-DEMAND ADENO-ASSOCIATED-VIRUS RELEASE FROM HYDROGEL MICROBEADS FOR GENE THERAPY

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

This paper reports ultrasound-triggered adeno-associated-virus (AAV) release from alginate hydrogel microbeads loaded into a gelatin hydrogel carrier to promote the transfection of cells. As a AAV carrier, caldium alginate hydrogel microbeads were centrifugally fabricated by ejecting a mixed pre-gel solution composed of sodium alginate, AAV and tungsten particles to CaCl2 solution to form ionically cross-linked instantly. For fixing of the beads, the microbeads were loaded into gelatin hydrogel carriers. The tungsten particles having high acoustic impedance enable the beads to have high sensitivity to ultrasound for improvement of the transfection. Our proposed ultrasound-triggered AAV releasing system would be an effective controlled release system for gene therapy.

PULSATILE FLOW ANALYSIS AT BRANCHED POINT IN ECM-BASED ENDOTHELIAL VASCULAR MODEL UNDER MECHANICAL STRETCH

Muramatsu J., Hashimoto M., Miura S., Onoe H.

This paper describes a stretchable extracellular matrix (ECM)-based in vitro endothelial vascular model with branched channels that can reproduce mechanically driven pulsatile flow. We imaged and analyzed the pulsatile flow at the branched points that are unique structures of blood vessels. The analysis revealed characteristic flow streams to the cultured endothelial cells near the branched points. We believe that our system would reveal the complex mechanical cellular response in blood vessels and enable the construction of mature in vitro vascular tissues.

GLUCOSE-MONITORING JANUS HYDROGEL MICROBEADS WITH COMPENSATION FUNCIONS FOR SURROUNDING PH ENVIRONMENT

Ando M., Tsuchiya M., Itai S., Heo Y.J., Onoe H.

This paper presents Janus microbeads for continuous fluorescent glucose monitoring with pH calibration. By using an UV- assisted centrifugal microfluidic device, the Janus hydrogel microbeads that consist of glucose sensor and pH sensor are easily fabricated. The microbead can calibrate the pH values of its surroundings and enables accurate measurements of glucose among various pH conditions. As a proof of concept, an accurate measurement of glucose concentration in the body-fluid-like solution using fluorescent intensities from glucose and pH sensor in microbeads was demonstrated. We believe that our fluorescent microbeads, with pH-calibration capacity, could be applied to fully implantable sensors for continuous glucose monitoring for diabetics.

CATHETER DELIVERY OF RADIOPAQUE CELL-ENCAPSULATED HYDROGEL MICROFIBERS FOR CELL THERAPY

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

This paper proposes a cell-encapsulated core-shell radiopaque hydrogel microfiber for in vivo cell therapy. The microfiber is composed of barium alginate gel containing zirconia particles in the shell and collagen gel encapsulating mesenchymal stem cells in the core. We confirmed that the cells in the fiber secrete paracrine factors to the outside of the fiber. Moreover, we demonstrated the improvement of the renal function of the rats by delivering the fiber into renal arteries. We believe that our fiber would treat incurable diseases including tissue degeneration.

Cell-encapsulated chitosan-collagen hydrogel hybrid nerve guidance conduit for peripheral nerve regeneration

Itai S., Suzuki K., Kurashina Y., Kimura H., Amemiya T., Sato K., Nakamura M., Onoe H.

Biomedical Microdevices (Biomedical Microdevices) 22 ( 4 ) 2020.12

ISSN 13872176

© 2020, Springer Science+Business Media, LLC, part of Springer Nature. Nerve guidance conduits (NGCs) composed of biocompatible polymers have been attracting attention as an alternative for autograft surgery in peripheral nerve regeneration. However, the nerve tissues repaired by NGCs often tend to be inadequate and lead to functional failure because of the lack of cellular supports. This paper presents a chitosan-collagen hydrogel conduit containing cells to induce peripheral nerve regeneration with cellular support. The conduit composed of two coaxial hydrogel layers of chitosan and collagen is simply made by molding and mechanical anchoring attachment with holes made on the hydrogel tube. A chitosan layer strengthens the conduit mechanically, and a collagen layer provides a scaffold for cells supporting the axonal extension. The conduits of different diameters (outer diameter approximately 2–4 mm) are fabricated. The conduit is bioabsorbable with lysozyme, and biocompatible even under bio absorption. In the neuron culture demonstration, the conduit containing Schwann cells induced the extension of the axon of neurons directed to the conduit. Our easily fabricated conduit could help the high-quality regeneration of peripheral nerves and contribute to the nerve repair surgery.

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Multiphoton photoreduction for dual-wavelength-light-driven shrinkage and actuation in hydrogel

Mizuguchi K., Nagano Y., Nishiyama H., Onoe H., Terakawa M.

Optical Materials Express (Optical Materials Express) 10 ( 8 ) 1931 - 1940 2020.08

© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement. The dual-wavelength-driven shrinkage of metal microstructures and hydrogel actuation are demonstrated by the fabrication of multi-metal microstructures in hydrogels by multiphoton photoreduction. Silver and gold microstructures were fabricated in a poly-Nisopropylacrylamide (PNIPAm) hydrogel. Because of the different optical resonances of the metals, wavelength-dependent shrinkage of metal microstructures was demonstrated concurrently with the volume change of the supporting hydrogel by light stimulation. Furthermore, the direction of actuation of the hydrogel was controlled by switching the wavelength of light stimulation. The results indicate the potential of multiphoton photoreduction for applications in light-driven optical components and micro-robots fabricated with soft materials.

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ECM-based microfluidic gradient generator for tunable surface environment by interstitial flow

Shimizu A., Goh W.H., Itai S., Karyappa R., Hashimoto M., Onoe H.

Biomicrofluidics (Biomicrofluidics) 14 ( 4 ) 2020.07

ISSN 19321058

© 2020 Author(s). We present an extracellular matrix (ECM)-based gradient generator that provides a culture surface with continuous chemical concentration gradients created by interstitial flow. The gelatin-based microchannels harboring gradient generators and in-channel micromixers were rapidly fabricated by sacrificial molding of a 3D-printed water-soluble sacrificial mold. When fluorescent dye solutions were introduced into the channel, the micromixers enhanced mixing of two solutions joined at the junction. Moreover, the concentration gradients generated in the channel diffused to the culture surface of the device through the interstitial space facilitated by the porous nature of the ECM. To check the functionality of the gradient generator for investigating cellular responses to chemical factors, we demonstrated that human umbilical vein endothelial cells cultured on the surface shrunk in response to the concentration gradient of histamine generated by interstitial flow from the microchannel. We believe that our device could be useful for the basic biological study of the cellular response to chemical stimuli and for the in vitro platform in drug testing.

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ECM-based microchannel for culturingin vitrovascular tissues with simultaneous perfusion and stretch

Shimizu A., Goh W.H., Itai S., Hashimoto M., Miura S., Onoe H.

Lab on a Chip (Lab on a Chip) 20 ( 11 ) 1917 - 1927 2020.06

ISSN 14730197

© The Royal Society of Chemistry 2020. We present an extracellular matrix (ECM)-based stretchable microfluidic system for culturingin vitrothree-dimensional (3D) vascular tissues, which mimicsin vivoblood vessels. Human umbilical vein endothelial cells (HUVECs) can be cultured under perfusion and stretch simultaneously with real-time imaging by our proposed system. Our ECM (transglutaminase (TG) cross-linked gelatin)-based microchannel was fabricated by dissolving water-soluble sacrificial polyvinyl alcohol (PVA) molds printed with a 3D printer. Flows in the microchannel were analyzed under perfusion and stretch. We demonstrated simultaneous perfusion and stretch of TG gelatin-based microchannels culturing HUVECs. We suggest that our TG gelatin-based stretchable microfluidic system proves to be a useful tool for understanding the mechanisms of vascular tissue formation and mechanotransduction.

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ECM-Based Gradient Generator for Tunable Surface Environment by Interstitial Flow

Shimizu A., Goh W.H., Karyappa R.B., Hashiomoto M., Onoe H.

ISSN 9781728135809

© 2020 IEEE. We present an extracellular matrix(ECM)-based microfluidic device that creates a spatial chemical gradient by interstitial flow. Cells located on surfaces of ECM, such as vascular and skin tissues, are influenced by external factors, including interstitial flows, but few methods have been available to recapitulate such conditions. To address this gap, we developed a microfluidic device that can expose cells cultured on the surface of ECM to a continuous concentration gradient created by the interstitial flow. With a 3D printed water-soluble sacrificial mold, we readily fabricated ECM-based microfluidic devices embedding micromixers and a gradient generator. ECM mimicked the permeability of in vivo environments. Our device will serve as a platform to study cellular phenomena occurring on the surface of ECM, such as vascularization and migration of cancer cells.

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Self-Folding Acute-Angle Origami Driven by Surface Bending Force

Uchida T., Yasuga H., Tachi T., Iwase E., Onoe H.

ISSN 9781728135809

© 2020 IEEE. This study describes a self-folding origami technique that enables acute angle folding by surface bending force for the first time. We patterned plus-shaped '+' carbon black patterns on a shrinking sheet and achieved self-folding of the 2D sheet quite sharply (more than 160°) trigger with infrared (IR) light irradiation. We investigated the folding angle of our sheet with '+' pattern comparing to the sheet with slit pattern on a crease, and confirmed that our sheet could be folded at larger folding angle than the sheet with slit pattern. Next, we examined the folding angle of the sheet that had '+' pattern with different width and found that the folding angle increased when the surface bending force increased. Finally, we demonstrated this '+' pattern to fabricate 3D word objects 'MEMS'. Our surface bending approach to Origami folding could open a new avenue to widen the variety and controllability of self-folding objects.

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PNIPAM/SWCNT-Based Hydrogel Micro-Gripper Driven by Infrared Light for Intravascular Surgery

Kuroda T., Onoe H.

ISSN 9781728135809

© 2020 IEEE. This paper describes an infrared (IR) light-responsive hydrogel micro-gripper for intravascular surgery. The hydrogel micro-gripper was fabricated by two-step photolithography that can design the motion of gripper. The gripping motion was controlled by IR exposure that was efficiently and selectively absorbed by the single walled carbon nanotubes (SWCNT) to generate heat. We confirmed the gripping speed in micro-scale gripper (width: sim 200 mumathrm{m}) was faster than that of the large scale (width: ∼2 mm). We demonstrated the hydrogel micro-gripper firmly gripped and kept holding a 200-mumathrm{m}-diameter bead with the chamber being shaken. We believe that our hydrogel micro-gripper can be applied for medical applications such as low-invasive medical practices because it could grip cells or foreign objects by being exposed to IR light irradiated from outside of the body.

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