Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

The lungs are important organs that play a critical role in the development of specific diseases, as well as responding to the effects of drugs, chemicals, and environmental pollutants. Due to the ethical concerns around animal testing, alternative methods have been sought which are more time-effective, do not pose ethical issues for animals, do not involve species differences, and provide easy investigation of the pathobiology of lung diseases. Several national and international organizations are working to accelerate the development and implementation of structurally and functionally complex tissue models as alternatives to animal testing, particularly for the lung. Unfortunately, to date, there is no lung tissue model that has been accepted by regulatory agencies for use in inhalation toxicology. This review discusses the challenges involved in developing a relevant lung tissue model derived from human cells such as cell lines, primary cells, and pluripotent stem cells. It also introduces examples of two-dimensional (2D) air-liquid interface and monocultured and co-cultured three-dimensional (3D) culture techniques, particularly organoid culture and 3D bioprinting. Furthermore, it reviews development of the lung on a chip model to mimic the microenvironment and physiological performance. The applications of lung tissue models in various studies, especially disease modeling, viral respiratory infection, and environmental toxicology will be also introduced. The development of a relevant lung tissue model is extremely important for standardizing and validation the in vitro models for inhalation toxicity and other studies in the future.

DOI： 10.1016/j.lfs.2023.122208

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DOI： 10.1186/s12576-023-00867-3

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DOI： 10.1186/s12576-023-00867-3

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DOI： 10.1186/s12576-022-00851-3

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Oxford University Press (OUP)  

Abstract

Background

Nitric oxide (NO), released from vascular endothelial cells in response to mechanical stimuli, regulates cardiac contractility and are also involved in the prevention of the development of cardiac hypertrophy.

Purpose

To establish an experimental system for live observation of NO release in response to mechanical stimuli on an organ chip.

Methods

Organ chips, which we used for the development of a heart-on-a-chip in the previous study [1], were used.

We seeded 300,000 human umbilical vein endothelial cells on a stretchable elastic membrane coated with Matrigel of a chip channel. Shear stress was applied to the cells by increasing flow rate of a peristaltic pump connected to the chip channel (Figure 1A). Pressure stimulus was applied by hydrostatic pressure. Stretch stimulus was applied by suction to the side ports of a chip using an electric syringe pump (Figure 1B). Cells were stained with 10 μM 4,5-diaminofluorescein diacetate for fluorescent live NO imaging.

Results

Monolayers of the endothelial cells formed intercellular junctions confirmed by CD31 staining (Figure 1C, yellow). Apparent permeability, which was measured by Texas red dye (MW 3000), was maintained at a low level of ∼3x10–6 cm/s until day 30, suggested the formation of robust intercellular junction.

When the endothelial cells were subjected to a pressure stimulus of 60 mmHg for 60 s, NO release was observed that lasted for >2 minutes (Figure 2A). A peak value of 1.46±1.08 (mean ± standard deviation) times the baseline was observed 271 s after the beginning of the pressure stimulus (n=251 cells). When the cells were subjected to a 1% stretch for 60 s, a peak value of 1.29±0.33 times the baseline was observed 105 s after the beginning of the stretch stimulus (Figure 2B). A shear stress of 0.01 dyn/cm2 hardly increased NO release (1.20±0.27 times the baseline, Figure 2C).

Conclusion

The system for live NO imaging in vascular endothelial cells in response to mechanical stimuli was established using organ-on-a-chip. The heart-on-a-chip with endothelial cells will be useful in elucidating the effects of mechanical stimulus such as hypertension on the contractile function and the remodeling of the heart.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science

DOI： 10.1093/eurheartj/ehac544.3027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

The error bars indicated in the figures in the article were standard deviation, although they should actually be standard errors of means. [Figure presented] [Figure presented] [Figure presented] [Figure presented] The authors would like to apologize for any inconvenience caused.

DOI： 10.1016/j.bbrc.2021.08.041

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Oxford University Press (OUP)  

Abstract

Background

The use of animal models in cardiovascular research is associated with two serious, intrinsic problems: inaccuracy in the extrapolation of data obtained from animals such as rodents due to different cardiac physiology and animal ethics.

Purpose

To develop an artificial human heart model for cardiovascular research using organ-on-a-chip technology and human cells

Methods

Organ chips made of silicone (polydimethylsiloxane) that have two microfluidic channels, a top channel and a bottom channel, separated by a 50-μm thick membrane with 7-μm pores hexagonally packed at 40-μm intervals (Figure 1), were used in this study.

We seeded 10,000 human umbilical vein endothelial cells on the membrane surface in the bottom channel to mimic the vasculature. Next, we seeded a mixture of 100,000 human-induced pluripotent stem cells (hiPSCs) and 50,000 human gingival fibroblasts on the membrane surface in the top channel (Figure 1). Human gingival fibroblasts facilitate cardiac differentiation of hiPSCs [1]. We performed the cardiac differentiation of hiPSCs using a previously described protocol [1]. Culture medium was perfused at a constant rate of 60 μl/h to maintain the culture.

Results

We observed spontaneous contraction of hiPSC-derived cardiomyocytes 20–26 days after the start of the differentiation protocol. Simultaneously, we conducted live intracellular calcium imaging using a fluorogenic calcium-sensitive dye, Cal-520 AM (5 μM in the culture medium). hiPSC-derived cardiomyocytes exhibited a periodic, coordinated pattern of calcium influx synchronised with their contraction under fluorescence microscopy (Figure 2A). Moreover, we found that the β-adrenergic agonist noradrenaline elevated the heart rate of hiPSC-derived cardiomyocytes on the organ chips in a dose-dependent manner (Figures 2A, B).

After observing the contraction and intracellular calcium influx of hiPSC-derived cardiomyocytes, we performed immunocytochemistry. Confocal microscopy indicated that the fluorescent signal obtained from anti-cardiac troponin T antibody staining in the top channel exhibited a typical striated pattern with 1.56±0.12-μm interval that reflected sarcomere structure (Figure 2C, yellow). Moreover, the fluorescent signal obtained from anti-CD31 antibody staining in the bottom channel exhibited a typical pattern at the boundary between cells, which is expected at the cell–cell junction of endothelial cells.

Conclusion

We developed a human heart-on-a-chip model that was confirmed by the functional response to noradrenaline and the histological evidence of sarcomere structure and vasculature, with a capability of live imaging. We expect that this model would be useful for examining the physiological function and for the pharmacological analysis of not only the normal heart but also the heart that reflects specific patient's pathophysiology using patient-derived hiPSCs.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science Figure 1Figure 2

DOI： 10.1093/eurheartj/ehab724.3190

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DOI： 10.1186/s12576-021-00809-x

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DOI： 10.1186/s12576-021-00809-x

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DOI： 10.1186/s12576-021-00809-x

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Gravity affects the function and maintenance of organs, such as bones, muscles, and the heart. Several studies have used DNA microarrays to identify genes with altered expressions in response to gravity. However, it is technically challenging to combine the results from various microarray datasets because of their different data structures. We hypothesized that it is possible to identify common changes in gene expression from the DNA microarray datasets obtained under various conditions and methods. In this study, we grouped homologous genes to perform a meta-analysis of multiple vascular endothelial cell and skeletal muscle datasets. According to the t-distributed stochastic neighbor embedding (t-SNE) analysis, the changes in the gene expression pattern in vascular endothelial cells formed specific clusters. We also identified candidate genes in endothelial cells that responded to gravity. Further, we exposed human umbilical vein endothelial cells (HUVEC) to simulated microgravity (SMG) using a clinostat and measured the expression levels of the candidate genes. Gene expression analysis using qRT-PCR revealed that the expression level of the prostaglandin (PG) transporter gene <italic>SLCO2A1</italic> decreased in response to microgravity, consistent with the meta-analysis of microarray datasets. Furthermore, the direction of gravity affected the expression level of <italic>SLCO2A1</italic>, buttressing the finding that its expression was affected by gravity. These results suggest that a meta-analysis of DNA microarray datasets may help identify new target genes previously overlooked in individual microarray analyses.

DOI： 10.3389/fcell.2021.689662

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DOI： 10.1016/j.bbrc.2021.03.077

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DOI： 10.1016/j.mex.2021.101404

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Lifestyle changes, such as overeating and underexercising, can increase the risk of prediabetes. Diabetes is one of the leading causes of atherosclerosis, and recently it became clear that the pathophysiology of atherosclerosis progresses even before the onset of diabetic symptoms. In addition to changes in platelets and leukocytes in the hyperglycemic state and damage to vascular endothelial cells, extracellular vesicles and microRNAs were found to be involved in the progression of prediabetes atherosclerosis. This review discusses the cellular and molecular mechanisms of these processes, with an intention to enable a comprehensive understanding of the pathophysiology of prediabetes and atherosclerosis.

DOI： 10.3390/ijms22084108

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Reactive oxygen species (ROS) plays a role in intracellular signal transduction under physiological conditions while also playing an essential role in diseases such as hypertension, ischemic heart disease, and diabetes, as well as in the process of aging. The influence of ROS has some influence on the frequent occurrence of cardiovascular diseases (CVD) in diabetic patients. In this review, we considered the pathophysiological relationship between diabetes and CVD from the perspective of ROS. In addition, considering organ damage due to ROS elevation during ischemia–reperfusion, we discussed heart and lung injuries. Furthermore, we have focused on the transient receptor potential (TRP) channels and L-type calcium channels as molecular targets for ROS in ROS-induced tissue damages and have discussed about the pathophysiological mechanism of the injury.

DOI： 10.3389/fcvm.2021.649785

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DOI： 10.1101/2021.03.31.437982

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A thrombus in a coronary artery causes ischemia, which eventually leads to myocardial infarction (MI) if not removed. However, removal generates reactive oxygen species (ROS), which causes ischemia–reperfusion (I/R) injury that damages the tissue and exacerbates the resulting MI. The mechanism of I/R injury is currently extensively understood. However, supplementation of exogenous antioxidants is ineffective against oxidative stress (OS). Enhancing the ability of endogenous antioxidants may be a more effective way to treat OS, and exosomes may play a role as targeted carriers. Exosomes are nanosized vesicles wrapped in biofilms which contain various complex RNAs and proteins. They are important intermediate carriers of intercellular communication and material exchange. In recent years, diagnosis and treatment with exosomes in cardiovascular diseases have gained considerable attention. Herein, we review the new findings of exosomes in the regulation of OS in coronary heart disease, discuss the possibility of exosomes as carriers for the targeted regulation of endogenous ROS generation, and compare the advantages of exosome therapy with those of stem-cell therapy. Finally, we explore several miRNAs found in exosomes against OS.

DOI： 10.3390/ijms22041729

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Gravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space.

DOI： 10.1038/s41526-020-00130-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

The error bars shown in Fig. 4 were standard deviation (SD), although they actually should be standard errors of means (SEM). The corrected figure is as below. The authors would like to apologise for any inconvenience caused.

DOI： 10.1016/j.mex.2021.101506

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DOI： 10.3791/61104

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Language：English   Publishing type：Research paper (scientific journal)  

The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1β and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.

DOI： 10.1016/j.yexcr.2019.111556

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DOI： 10.1016/j.bbrc.2019.09.119

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The application of mechanical stimuli to cells often induce increases in intracellular calcium, affecting the regulation of a variety of cell functions. Although the mechanism of mechanotransduction-induced calcium increases has not been fully resolved, the involvement of mechanosensitive ion channels in the plasma membrane and the endoplasmic reticulum has been reported. Here, we demonstrate that voltage-gated L-type calcium channels play a critical role in the mechanosensitive calcium response in H9c2 rat cardiomyocytes. The intracellular calcium level in H9c2 cells increased in a reproducible dose-dependent manner in response to uniaxial stretching. The stretch-activated calcium response (SICR) completely disappeared in calcium-free medium, whereas thapsigargin and cyclopiazonic acid, inhibitors of sarcoendoplasmic reticulum calcium ATPase, partially reduced the SICR. These findings suggest that both calcium influx across the cell membrane and calcium release from the sarcoendoplasmic reticulum are involved in the SICR. Nifedipine, diltiazem, and verapamil, inhibitors of L-type calcium channels, reduced the SICR in a dose-dependent manner. Furthermore, small interfering RNA against the L-type calcium channel α1c subunit diminished the SICR dramatically. Nifedipine also diminished the mechanosensitivity of Langendorff-perfused rat heart. These results suggest that the SICR in H9c2 cardiomyocytes involves the activation of L-type calcium channels and subsequent calcium release from the sarcoendoplasmic reticulum.

DOI： 10.1016/j.ceca.2019.02.008

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DOI： 10.1016/j.bbrc.2018.07.116

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DOI： 10.3390/cells7060062

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Tokyo  

When a cardiac muscle is held in a stretched position, its [Ca2+] transient increases slowly over several minutes in a process known as stress-induced slow increase in intracellular Ca2+ concentration ([Ca2+]i) (SSC). Transient receptor potential canonical (TRPC) 3 forms a non-selective cation channel regulated by the angiotensin II type 1 receptor (AT1R). In this study, we investigated the role of TRPC3 in the SSC. Isolated mouse ventricular myocytes were electrically stimulated and subjected to sustained stretch. An AT1R blocker, a phospholipase C inhibitor, and a TRPC3 inhibitor suppressed the SSC. These inhibitors also abolished the observed SSC-like slow increase in [Ca2+]i induced by angiotensin II, instead of stretch. Furthermore, the SSC was not observed in TRPC3 knockout mice. Simulation and immunohistochemical studies suggest that sarcolemmal TRPC3 is responsible for the SSC. These results indicate that sarcolemmal TRPC3, regulated by AT1R, causes the SSC.

DOI： 10.1007/s12576-016-0519-3

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DOI： 10.1007/s12576-018-0610-z

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DOI： 10.1007/s12576-018-0610-z

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DOI： 10.1007/s12576-018-0610-z

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DOI： 10.1093/eurheartj/ehx501.45

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Long-term habitation in space leads to physiological alterations such as bone loss, muscle atrophy, and cardiovascular deconditioning. Two predominant factors-namely space radiation and microgravity-have a crucial impact on oxidative stress in living organisms. Oxidative stress is also involved in the aging process, and plays important roles in the development of cardiovascular diseases including hypertension, left ventricular hypertrophy, and myocardial infarction. Here, we discuss the effects of space radiation, microgravity, and a combination of these two factors on oxidative stress. Future research may facilitate safer living in space by reducing the adverse effects of oxidative stress.

DOI： 10.3390/ijms18071426

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

DOI： 10.1016/j.bpj.2016.11.1471

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Language：Japanese   Publisher：(一社)日本生理学会  

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DOI： 10.1007/bf03405812

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DOI： 10.1007/bf03405812

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Part of collection (book)   Publisher：wiley  

Long-term exposure to pressure/volume overload of the heart due to hypertension or aortic stenosis induces pathological hypertrophy and remodeling, and eventually causes heart failure. This process accompanies pathological phenomena such as inflammation, apoptosis, and arrhythmia. On the other hand, physical exercise promotes physiological hypertrophy and remodeling of the heart, thereby enhancing cardiac performance. Interestingly, recent research has suggested that enhancement of heart function is a result not only of physiological hypertrophy of pre-existing cardiomyocytes, but also of cardiac stem cell activation and myocyte formation. Furthermore, exercise improves exacerbated cardiac function in heart diseases, including dilated cardiomyopathy (DCM) and myocardial infarction (MI). The suggested mechanisms of action of exercise remedies for these diseases include angiogenesis, cardiac resynchronization, and inhibition of inflammatory cytokines. However, excessive exercise can cause arrhythmogenic remodeling and sudden death. In this chapter, we elaborate the mechanisms of hypertrophy and remodeling under physiological and pathological conditions and discuss future directions for coping with pathological hypertrophy and remodeling.

DOI： 10.1002/9781118966174.ch11

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This article describes how physical forces contribute to development, physiology, and pathology of vascular cells, focusing on endothelial cells and vascular smooth muscle cells. Based on these basic understandings of the mechanobiology, we discuss mechanomedicine, an application of the mechanobiology to medicine. Basic knowledge about cellular responses, such as cellular signal transduction pathway, gene expression, and cytoskeletal remodeling, to mechanical stimuli is important for understanding the pathology of vascular diseases including atherosclerosis. Introducing the knowledge of the vascular mechanobiology will not only contribute to the development of regenerative medicine using pluripotent stem cells but also provide a way to prevent diseases caused by thromboembolisms, such as myocardial and cerebral infarctions.

DOI： 10.1007/978-4-431-54801-0_15

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Institute of Mathematical Sciences  

Cell surface receptors are involved in numerous important biological processes including embryogenesis, tissue differentiation, and cellular homeostasis. Among them, mechanosensitive ion channels play an essential role in cellular functions of every cell including neurons, cardiomyocytes, and osteocytes. Here, we discuss types, roles, structures, and biophysical factors that affect the functions of mechanosensitive ion channels.

DOI： 10.3934/biophy.2016.1.63

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

All previous reports concerning the effect of stretch on cultured skin cells dealt with experiments on epidermal keratinocytes or dermal fibroblasts alone. The aim of the present study was to develop a system that allows application of stretch stimuli to human skin equivalents (HSEs), prepared by coculturing of these two types of cells. In addition, this study aimed to analyze the effect of a stretch on keratinization of the epidermis and on the basement membrane. HSEs were prepared in a gutter-like structure created with a porous silicone sheet in a silicone chamber. After 5-day stimulation with stretching, HSEs were analyzed histologically and immunohistologically. Stretch-stimulated HSEs had a thicker epidermal layer and expressed significantly greater levels of laminin 5 and collagen IV/VII in the basal layer compared with HSEs not subjected to stretch stimulation. Transmission electron microscopy revealed that the structure of the basement membrane was more developed in HSEs subjected to stretching. Our model may be relevant for extrapolating the effect of a stretch on the skin in a state similar to an in vivo system. This experimental system may be useful for analysis of the effects of stretch stimuli on skin properties and wound healing and is also expected to be applicable to an in vitro model of a hypertrophic scar in the future.

DOI： 10.1371/journal.pone.0141989

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Ischemic heart disease still remains the most common cause of cardiac death. During ischemia-reperfusion (I/R), reactive oxygen species (ROS) are produced in excess in cardiac tissue, where they induce cell death. Our previous study showed that 9-phenanthrol (9-Phe), a specific inhibitor of the TRPM4 channel, preserves cardiac contractile function and protects the heart from I/R injury-related infarction in the excised rat heart. Accordingly, we hypothesized that TRPM4 channels are involved in the 9-Phe-mediated cardioprotection against ROS-induced injury. In rats, intravenous 9-Phe mitigated the development of myocardial infarction caused by the occlusion of the left anterior descending artery. Immunohistochemical analysis indicated that TRPM4 proteins are expressed in ventricular myocytes susceptible to I/R injury. Hydrogen peroxide (H2O2) is among the main ROS overproduced during I/R. In the cardiomyocyte cell line H9c2, pretreatment with 9-Phe prevented cell death induced by conditions mimicking I/R, namely 200 mu M H2O2 and hypoxia-reoxygenation. Gene silencing of TRPM4 preserved the viability of H9c2 cardiomyocytes exposed to 200 mu M H2O2. These results suggest that the cardioprotective effects of 9-Phe are mediated through the inhibition of the TRPM4 channels.

DOI： 10.1371/journal.pone.0121703

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

DOI： 10.1016/j.bpj.2014.11.3189

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DOI： 10.1007/bf03405845

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Institute of Mathematical Sciences  

Coarse-grained molecular dynamics (CGMD) simulations are increasingly being used to analyze the behaviors of biological systems. When appropriately used, CGMD can simulate the behaviors of molecular systems several hundred times faster than elaborate all-atom molecular dynamics simulations with similar accuracy. CGMD parameters for lipids, proteins, nucleic acids, and some artificial substances such as carbon nanotubes have been suggested. Here we briefly discuss a method for CGMD system configuration and the types of analysis and perturbations that can be performed with CGMD simulations. We also describe specific examples to show how CGMD simulations have been applied to various situations, and then describe experimental results that were used to validate the simulation results. CGMD simulations are applicable to resolving problems for various biological systems.

DOI： 10.3934/biophy.2014.1.1

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Authorship：Lead author   Language：Japanese  

DOI： 10.11392/jsao.43.206

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DOI： 10.1007/BF03405834

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Medical and Biological Engineering  

DOI： 10.11239/jsmbe.51.R-14

CiNii Article

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DOI： 10.11239/jsmbe.51.R-149

CiNii Article

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Despite efforts to elucidate its pathophysiology, ischemia-reperfusion injury lacks an effective preventative intervention. Because transient receptor potential cation channel subfamily M member 4 (TRPM4) is functionally expressed by many cell types in the cardiovascular system and is involved in the pathogenesis of various cardiovascular diseases, we decided to assess its suitability as a target of therapy. Thus, the aim of this study was to examine the possible cardioprotective effect of 9-phenanthrol, a specific inhibitor of TRPM4. Isolated Langendorff-perfused rat hearts were pretreated with Krebs-Henseleit (K-H) solution (control), 9-phenanthrol, or 5-hydroxydecanoate (5-HD, a blocker of the ATP-sensitive potassium channel) and then subjected to global ischemia followed by reperfusion with the K-H solution. To evaluate the extent of heart damage, lactate dehydrogenase (LDH) activity in the effluent solution was measured, and the size of infarcted area of the heart was measured by 2,3,5-triphenyltetrazolium chloride staining. In controls, cardiac contractility decreased, and LDH activity and the infarcted area size increased. In contrast, in hearts pretreated with 9-phenanthrol, contractile function recovered dramatically, and the infarcted area size significantly decreased. The cardioprotective effects of 9-phenanthrol was not completely blocked by 5-HD. These findings show that 9-phenanthrol exerts a cardioprotective effect against ischemia in the isolated rat heart and suggest that its mechanism of action is largely independent of ATP-sensitive potassium channels.

DOI： 10.1371/journal.pone.0070587

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Mechanosensitivity is essential for heart function just as for all other cells and organs in the body, and it is involved in both normal physiology and diseases processes of the cardiovascular system. In this review, we have outlined the relationship between mechanosensitivity and heart physiology, including the FrankStarling law of the heart and mechanoelectric feedback. We then focused on molecules involved in mechanotransduction, particularly mechanosensitive ion channels. We have also discussed the involvement of mechanosensitivity in heart diseases, such as arrhythmias, hypertrophy and ischaemic heart disease. Finally, mechanobiology in cardiogenesis is described with regard to regenerative medicine.

DOI： 10.1111/jcmm.12027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Manual palpation or pressure stimulation is often used for pain sensitivity assessment. The aim of the current study was to define a method for investigating the relation between pressure pain sensitivity and pressure propagation in soft or harder muscles. Three-dimensional finite-element computer-models were developed to simulate the tissue stress and strain distribution during pressure stimulation on the tibialis anterior and gastrocnemius muscles. Four cases were modelled representing females and males who were trained and untrained. The model geometry was based on MR images of the lower leg during pressure stimulation. Stress and strain were extracted from the models at pressure intensity levels equivalent to the pressure pain threshold. The principal strain peaked in the adipose tissue at 0.30 and 0.14 for stimulation on the gastrocnemius and tibialis anterior muscle, respectively. The principal strain in the muscle was higher for four models of the stimulation on the gastrocnemius muscle (0.22-0.30) compared with the four models of stimulation on the tibialis anterior muscle (0.11-0.14). Average pressure pain thresholds were significantly lower for the tibialis anterior compared with the gastrocnemius muscle (319 vs. 432 kPa) These data show different pressure propagation profiles in soft and hard muscle at the same pressure pain sensation level. This new approach is relevant as the clinical routine assesses all muscles equally. This results in a different exposure to pressure in relation to the muscle evaluated which may affect the outcome of the examination.

DOI： 10.1007/s11517-012-0974-9

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The heart is an organ that is exposed to extreme dynamic mechanical stimuli. From birth till death, the heart indefinitely repeats periodic contraction and dilation, i.e., shortening and elongation of cardiomyocytes. Mechanical stretch elicits a change in heart rate and may cause arrhythmia if it is excessive. Thus, mechanosensitivity is crucial to heart function. The molecule that is substantially involved in mechanosensitivity is a stretch-activated ion channel. Among several ion channels believed to be activated by stretch in the heart, the stretch-activated K-Ca (SAKCA) channel, a member of the group of large conductance (Big Potassium, BK) channels, shows a mechanosensitive (MS) response to membrane stretch. As BK channels respond to voltage and intracellular calcium concentration with large conductance, they are considered to be involved in repolarization after depolarization. Some BK channels are known to be activated by stretch and are expressed in a number of cells, including human osteoblasts and guinea pig intestinal neurons. The SAKCA channel was found to be sensitive to stretch in the chick heart. Given that the cardiomyocyte is unremittingly exposed to contraction and dilation and that it generates action potential and its contractility is modulated by intracellular calcium concentration, the SAKCA channel, which is dependent voltage and calcium, may be involved in action potential generation. It was recently reported that a BK channel is involved in the modulation of heart rate in the mouse. Further studies regarding the role of MS BK channels, including SAKCA, in the modulation of heart rate and contractility are expected. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.pbiomolbio.2012.08.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE INC MEDICAL PUBLISHERS  

DOI： 10.1016/j.cardfail.2012.08.116

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Ovid Technologies (Wolters Kluwer Health)  

DOI： 10.1097/01.hjh.0000420766.99570.ae

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Skin pain and muscle pain are categorically distinct from each other. While skin pain is a sharp, spatially localized sensation, muscle pain is a dull, poorly localized and more unpleasant one. We hypothesized that there are specific brain regions preferentially activated by muscle pain compared to skin pain. To test this hypothesis, brain responses were recorded from 13 normal male subjects in response to repeated painful electrical stimulation of the muscle and skin of the left leg, using 3-T magnetic resonance imaging scanner. The common brain regions that responded to painful stimulations of both skin and muscle were the thalamus, anterior cingulate cortex, bilateral insula, contralateral primary and secondary somatosensory cortices, and ipsilateral cerebellum. Brain regions specifically activated by muscle stimulation were the midbrain, bilateral amygdala, caudate, orbitofrontal cortex, hippocampus, parahippocampus and superior temporal pole, most of which are related to emotion. Regions except the midbrain showed contralateral preference. These results suggest that dull sensation, which is characteristic of muscular pain, is related with processing in these brain regions. (C) 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2011.04.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

Continued exposure of endothelial cells to mechanical/shear stress elicits the unfolded protein response (UPR), which enhances intracellular homeostasis and protect cells against the accumulation of improperly folded proteins. Cells commit to apoptosis when subjected to continuous and high endoplasmic reticulum (ER) stress unless homeostasis is maintained. It is unknown how endothelial cells differentially regulate the UPR. Here we show that a novel Girdin family protein, Gipie (78 kDa glucose-regulated protein [GRP78]-interacting protein induced by ER stress), is expressed in endothelial cells, where it interacts with GRP78, a master regulator of the UPR. Gipie stabilizes the interaction between GRP78 and the ER stress sensor inositol-requiring protein 1 (IRE1) at the ER, leading to the attenuation of IRE1-induced c-Jun N-terminal kinase (JNK) activation. Gipie expression is induced upon ER stress and suppresses the IRE1-JNK pathway and ER stress-induced apoptosis. Furthermore we found that Gipie expression is up-regulated in the neointima of carotid arteries after balloon injury in a rat model that is known to result in the induction of the UPR. Thus our data indicate that Gipie/GRP78 interaction controls the IRE1-JNK signaling pathway. That interaction appears to protect endothelial cells against ER stress-induced apoptosis in pathological contexts such as atherosclerosis and vascular endothelial dysfunction.

DOI： 10.1091/mbc.E10-08-0724

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

DOI： 10.1016/j.cbpb.2006.10.090

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Language：Japanese  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INFORMA HEALTHCARE  

Transcutaneous pressure with pressure probes of arbitrary diameters have been commonly used for measuring the threshold and magnitude of muscle pain, yet this procedure lacks scientific validation. To examine the valid probe dimensions, we conducted physiological experiments using 34 human subjects. Pin-prick pain, pressure pain threshold (PPT) to pressure probes of various diameters, heat pain threshold, and electrical pain threshold of deep tissues were measured before and after application of surface lidocaine anesthesia to the skin surface over the brachioradial muscle in a double-blinded manner. The anesthesia neither affected PPT with larger probes (diameters: 1.6 and 15 mm) nor increased electric pain threshold of deep structures, whereas it diminished pain count in pin-prick test and PPT with a 1.0 mm diameter probe, suggesting that mechanical pain thresholds measured with 1.6 and 15 mm probes reflect the pain threshold of deep tissues, possibly muscle. Pain thresholds to heat did not change after application of the anesthesia. These results suggest that larger pressure probes can give a better estimation of muscular pain threshold.

DOI： 10.1080/08990220500420475

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

To examine the effects of change in meteorological parameters on pain-related behaviors in a simulated arthritic condition, rats with an injection of complete Freund's adjuvant into the tibio-tarsal joint were exposed to low barometric pressure (20 mmHg below the natural atmospheric pressure) and low ambient temperature (7 degreesC lower than 22 degreesC) in a climate-controlled room. When the arthritic rats were exposed to these environments, the already increased number of hindpaw withdrawals in response to noxious mechanical stimulation (hyperalgesia) was further increased, and a hindpaw withdrawal response to innocuous mechanical stimulation (allodynia) began to occur. Such exposures did not influence any of the pain-related behaviors of the control rats. These results show that lowering barometric pressure and ambient temperature within the range of natural environmental fluctuation intensify pain in arthritic rats. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2003.09.057

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：INT ASSOC STUDY PAIN (IASP) PRESS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

Cold allodynia is an annoying symptom in conditions of chronic inflammation such as rheumatoid arthritis. To examine whether primary afferent nerve activities are changed in association with hypersensitivity to cold, we recorded single nerve activities from the sural nerve in persistently inflamed rats in vivo. Inflammation was induced by an injection of complete Freund's adjuvant (CFA) solution into the tibio-tarsal joint. Inflamed rats showed an increased number of paw shakes to paw immersion in 25degreesC water (pre-inflammation: 1.15 +/- 0.58, 2-week inflammation: 4.70 +/- 1.15). We also recorded cutaneous C-fiber activities under pentobarbital anesthesia and studied their responses to thermal and mechanical stimuli. The response of C-low threshold mechanoreceptors to cooling (total discharges between 27 and 23degreesC) increased 1.8-fold (control group: 5.17 +/- 1.04 impulses, inflamed group: 9.38 +/- 1.47 impulses). In addition, the proportion of C-nociceptor units responding to cold down to 2degreesC was significantly greater in the inflamed group (9 out of 18 units; threshold: 10.0 +/- 2.6degreesC) than in the intact group (1 out of 14 units; threshold: 4.0degreesC). These results suggest that the facilitated responses of these primary afferents are associated with cold hypersensitivity in chronically inflamed conditions. (C) 2003 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2003.08.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nagoya University  

DOI： 10.18999/envm.46.122

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Other Link： http://search.jamas.or.jp/link/ui/2003221359

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nagoya University  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Okayama Medical Association  

DOI： 10.4044/joma.128.61

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Other Link： https://jlc.jst.go.jp/DN/JLC/20022537940?from=CiNii

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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DOI： 10.11477/mf.2425100317

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Language：Japanese   Publishing type：Internal/External technical report, pre-print, etc.   Publisher：名古屋大学  

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Publisher：PHYSIOLOGICAL SOCIETY OF JAPAN  

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Event date： 2021.8.27 - 2021.8.30

Language：English   Presentation type：Poster presentation  

Venue：Online  

Background
The use of animal models in cardiovascular research is associated with two serious, intrinsic problems:
inaccuracy in the extrapolation of data obtained from animals such as rodents due to different cardiac physiology
and animal ethics.
Purpose
To develop an artificial human heart model for cardiovascular research using organ-on-a-chip technology and
human cells
Methods
Organ chips made of silicone (polydimethylsiloxane) that have two microfluidic channels, a top channel and a
bottom channel, separated by a 50-μm thick membrane with 7-μm pores hexagonally packed at 40-μm intervals
(Figure 1), were used in this study.
We seeded 10,000 human umbilical vein endothelial cells on the membrane surface in the bottom channel to
mimic the vasculature. Next, we seeded a mixture of 100,000 human-induced pluripotent stem cells (hiPSCs) and
50,000 human gingival fibroblasts on the membrane surface in the top channel (Figure 1). Human gingival
fibroblasts facilitate cardiac differentiation of hiPSCs [1]. We performed the cardiac differentiation of hiPSCs
using a previously described protocol [1]. Culture medium was perfused at a constant rate of 60 μl/h to maintain
the culture.
Results
We observed spontaneous contraction of hiPSC-derived cardiomyocytes 20–26 days after the start of the
differentiation protocol. Simultaneously, we conducted live intracellular calcium imaging using a fluorogenic
calcium-sensitive dye, Cal-520 AM (5 μM in the culture medium). hiPSC-derived cardiomyocytes exhibited a
periodic, coordinated pattern of calcium influx synchronised with their contraction under fluorescence
microscopy (Figure 2A). Moreover, we found that the β-adrenergic agonist noradrenaline elevated the heart rate
of hiPSC-derived cardiomyocytes on the organ chips in a dose-dependent manner (Figures 2A, B).
After observing the contraction and intracellular calcium influx of hiPSC-derived cardiomyocytes, we performed
immunocytochemistry. Confocal microscopy indicated that the fluorescent signal obtained from anti-cardiac
troponin T antibody staining in the top channel exhibited a typical striated pattern with 1.56 ± 0.12-μm interval
that reflected sarcomere structure (Figure 2C, yellow). Moreover, the fluorescent signal obtained from anti-CD31
antibody staining in the bottom channel exhibited a typical pattern at the boundary between cells, which is
expected at the cell–cell junction of endothelial cells.
Conclusion
We developed a human heart-on-a-chip model that was confirmed by the functional response to noradrenaline
and the histological evidence of sarcomere structure and vasculature, with a capability of live imaging. We expect
that this model would be useful for examining the physiological function and for the pharmacological analysis of
not only the normal heart but also the heart that reflects specific patient’s pathophysiology using patientderived
hiPSCs.

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Event date： 2021.7.2 - 2021.7.4

Language：English   Presentation type：Oral presentation (invited, special)  

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Event date： 2020.10.27 - 2020.10.30

Presentation type：Symposium, workshop panel (nominated)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：Singapore  

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Event date： 2025.3.17 - 2025.3.19

Language：English   Presentation type：Oral presentation (invited, special)  

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Event date： 2025.3.17 - 2025.3.19

Language：English   Presentation type：Oral presentation (invited, special)  

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Event date： 2025.3.7 - 2025.3.8

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Event date： 2025.3.4 - 2025.3.5

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Misasa, Japan  

As space travel moves beyond professional astronauts to include researchers, tourists, and eventually settlers on the Moon and Mars, understanding how the human body adapts to space environments has never been more important. Whether for a short suborbital flight or a long-term mission to another planet, microgravity and space radiation pose unique challenges to human health. In weightlessness, bodily fluids shift upwards, causing swollen faces and sinus congestion, muscles weaken from disuse, bones lose density at an accelerated rate, and even vision can deteriorate. Over time, the heart and immune system also undergo significant changes, raising concerns for long-duration space habitation.

Why does this happen? Gravity is a fundamental force that has shaped human biology for millions of years. Without it, our cells, tissues, and organs behave differently—muscle fibers shrink, bone cells stop regenerating properly, and even gene expression is altered. Interestingly, many of these effects resemble aging on Earth, making space an extreme but valuable environment for studying human physiology.

This talk will explore how spaceflight affects the human body and what it means for the future of space exploration. As more people prepare to live and work beyond Earth, we must develop strategies to keep them healthy, from artificial gravity solutions to personalized countermeasures. Understanding how humans can thrive in space is not just a challenge for astronauts—it’s a question that will define the future of human expansion beyond our planet.

Reference: [1] Takahashi K, et al. (2021) NPJ Microgravity, 8;7(1):2. [2] Liang Y, Wang M, Liu Y, Wang C, Takahashi K, Naruse K. (2021) Front Cell Dev Biol, 9:689662.

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Event date： 2024.3.28 - 2024.3.30

Language：English   Presentation type：Poster presentation  

Venue：Kokura  

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Event date： 2024.1.30 - 2024.2.2

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2023.11.17 - 2023.11.18

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：Tokushima  

Background: Vascular endothelial cells are constantly exposed to mechanical stressors such as blood pressure, vascular expansion, and shear stress due to blood flow. Nitric oxide (NO) released from endothelial cells in response to mechanical stress plays a role in regulating cardiac contractility and preventing the progression of myocardial hypertrophy. This study aimed to establish an experimental system for live imaging of mechanical stress-responsive NO release.

Methods: Human umbilical vein endothelial cells were seeded on an elastic membrane of a microfluidic chip with stretchable properties. Shear stress was applied to the cells by increasing the flow rate of the peristaltic pump connected to the fluid delivery tubing (Figure A). Pressure stimulation was applied through hydrostatic pressure. Stretch stimulation was applied to the cultured cells on the elastic membrane (Figure B). Cells were administered 10 μM of 4,5-diaminofluorescein diacetate, and fluorescence-based live NO imaging was performed.

Results: Immunostaining for the intercellular adhesion marker CD31 revealed the formation of robust intercellular adhesions (Figure C). Permeability assay using dextran (molecular weight 3,000) indicated that the apparent permeability, an indicator of barrier function, was maintained at a low value of approximately 3 × 10-6 cm/s for 30 days. This suggested that vascular endothelial cells on the microfluidic chip possess robust barrier function. When vascular endothelial cells were subjected to a pressure stimulus of 60 mmHg for 60 seconds, the peak NO concentration was observed 271 seconds after the start of the pressure stimulus, with NO release observed for over 2 minutes. Subsequently, when endothelial cells were subjected to a 1% stretch stimulus for 60 seconds, a peak value 1.29 ± 0.33 times higher than the baseline was observed 105 seconds after the start of the stretch stimulus. Application of 0.01 dyn/cm2 shear stress to the endothelial cells did not result in a significant increase in NO release.

Conclusion: A system for live imaging of mechanical stress-responsive NO release in vascular endothelial cells using an organ-on-a-chip has been established. By applying this technology to heart-on-a-chip systems, it is expected to elucidate the effects of mechanical stimuli, such as high blood pressure, on cardiac contractile function and remodeling.

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Event date： 2023.10.28 - 2023.10.29

Language：English   Presentation type：Oral presentation (general)  

Venue：Okayama  

Pulmonary fibrosis is a severe lung condition characterized by lung scarring and thickening, resulting in impaired breathing for affected individuals. While animal models have been commonly employed in research, the use of human cell-based models is essential for the development of more effective therapies for pulmonary fibrosis. In our efforts to establish a lung fibrosis model, we utilized human lung epithelial cell line NCI-H441 and human gingival fibroblasts (HGFs). We induced fibrosis-like changes by administering transforming growth factor beta-1 (TGF-β1). Morphological alterations were observed in HGFs under a microscope, although no such changes were seen in NCI-H441. Subsequently, we extracted mRNA and conducted RT-PCR to assess fibrosis marker expression in both cell types. Our analysis confirmed that the α-SMA gene expression increased in a dose-dependent manner in HGFs following TGF-β1 treatment, whereas NCI-H441 exhibited significant upregulation of fibrosis marker genes, including slug and vimentin. To better replicate the lung's physiological structure, we employed transwell culture systems. Human umbilical vein endothelial cells were seeded on the basolateral side. NCI-H441 and HGFs were then seeded on the apical side of the transwell. Upon reaching full confluence, the culture medium was replaced with TGF-β1-containing medium. After two days of treatment, we simultaneously extracted mRNAs from NCI-H441 and HGFs on the apical side and conducted RT-PCR analysis. Our findings revealed that the expression of fibrosis marker genes, including α-SMA, Slug, and Vimentin, tended to increase in response to TGF-β1. Additionally, we endeavored to create an air-liquid interface (ALI) within the lung alveoli by removing the culture medium on the apical side. Even two days later, confocal microscopy confirmed the uptake of intracellular calcium indicators, indicating successful ALI formation. Immunostaining for PECAM-1, an endothelial junction marker, demonstrated robust intercellular junction integrity among endothelial cells. We are committed to further refining this human triculture model to advance our contributions to the development of treatments for pulmonary fibrosis.

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Event date： 2023.9.8 - 2023.9.9

Language：English   Presentation type：Poster presentation  

Venue：Kumamoto  

Vascular endothelial cells play crucial roles in vasoregulation and atherosclerosis pathogenesis. Traditional 2D or static cell culture models limit morphology and functionality of endothelial cells. To mimic the dynamic microenvironment, we seeded human umbilical vein endothelial cells (HUVECs) into microfluidic organ chips. Assessing barrier function with a dextran permeability assay, we evaluated the impact of shear stress on vascular permeability at different flow rates. Optimized flow rate (80 μl/hr) enhanced barrier function, evidenced by minimized apparent permeability. HUVECs aligned parallel to flow on the microfluidic channel. We have established a method to culture highly barrier-functional endothelium with a similar orientation to in vivo blood vessels within microfluidic channels. This method holds promise for application in organ chips.

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Event date： 2023.3.14 - 2023.3.16

Language：English   Presentation type：Poster presentation  

Venue：Kyoto  

Pulmonary fibrosis is a severe lung disease causing scarring and thickening of the lungs, which make breathing difficult. Although animal models are often used to study lung fibrosis, models using human cells are necessary to develop better therapeutic strategies. To develop a human lung fibrosis model, NCI-H441 lung epithelial cells and human gingival fibroblasts (HGF) were first monocultured. The cells were treated with transforming growth factor beta-1 (TGF-β1) to induce fibrosis-like changes. TGF-β1 treatment increased the area of adhesion to the culture plate of fibroblasts, but not the lung epithelial cells. Gene expression analysis of fibrosis markers using quantitative reverse transcribed polymerase chain reaction (qRT-PCR) revealed that alpha smooth muscle actin (α-SMA) expression increased with increasing doses of TGF-β1 in HGF, but not in NCI-H441 cells. In contrast, expression of fibrosis marker genes, including Slug and Vimentin, increased in a dose-dependent manner in NCI-H441 cells. Transwells were used to recapitulate lung structure. Human umbilical vein endothelial cells were seeded on the basolateral side of the transwells, which were coated with Matrigel to facilitate cell adhesion. NCI-H441 cells and fibroblasts were seeded on the apical side of the transwells. After the cells reached 100% confluency, cells were treated with TGF-β1 for 2 days, and NCI-H441 and HGF were collected from the apical side of the transwells. The expression of fibrosis marker genes, including α-SMA, Slug, and Vimentin, were measured using qRT-PCR. These three marker genes tended to increase in response to TGF-β1. To achieve an air–liquid interface (ALI), similar to lung alveoli, the medium was removed from the apical side of the transwells. Uptake of an intracellular calcium indicator was observed under a confocal microscope even two days after removal of the medium, indicating the successful formation of an ALI. Additionally, immunostaining for PECAM-1 indicated high integrity of the intercellular junctions between the endothelial cells. We will further develop this human triculture model to facilitate the development of treatments for pulmonary fibrosis.

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Event date： 2023.3.14 - 2023.3.16

Language：English   Presentation type：Poster presentation  

Venue：Kyoto  

Cardiovascular diseases have always posed a serious threat to mankind. With the advent of pluripotent stem cell technology, the pathophysiology of human heart diseases can now be mimicked using tissue culture methods, including trans-well and microfluidic chip models. In this study, we developed a tri-culture model comprising human gingival fibroblasts (HGFs), human umbilical vein endothelial cells (HUVECs), and induced pluripotent stem cells (iPSCs) using both trans-wells and microfluidic chips. We seeded iPSCs and HGFs onto the top channel of a chip and the apical side of a trans-well, whereas HUVECs were seeded onto the bottom channel of the chip and the basolateral side of the trans-well. After iPSCs were differentiated into cardiomyocytes in each model, their contractility was compared. Interestingly, cardiomyocytes in the chip model exhibited spontaneous contraction as early as on day 16 post-commencement of differentiation whereas those in the trans-well model showed contractions on day 23. Moreover, 33.3% spontaneous contraction was observed in the chip model compared with 11% in the trans-well model. Fluorescence-activated cell sorting revealed that 12.6% of the cells collected from the top channels in the chip model expressed cardiac troponin T, a specific cardiomyocyte marker. Our study suggests that the continuous supply of medium to the cells may be an important factor governing the differentiation of iPSCs into cardiomyocytes.

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Event date： 2022.12.8 - 2022.12.10

Language：English   Presentation type：Poster presentation  

Venue：Los Angeles  

Drug pharmacology and toxicology tests using laboratory animals have limited applicability to humans and pose a considerable ethical dilemma. These problems can be resolved by developing an organ-on-a-chip technology that realistically simulates human organ function. We developed a heart-on-a-chip that contains fibroblasts similar to those of actual hearts and that also includes vascular endothelial layers that respond to pressure, stretch, and shear stress caused by blood flow. The heart chip consists of a myocardial channel that forms a co-culture system of human induced pluripotent stem cell-derived cardiomyocytes, human fibroblasts, and a vascular channel that forms a vascular endothelial cell layer separated by a 100 μm-thick permeable membrane. The cells in the myocardial channel spontaneously contracted without electrical stimulation and exhibited a dose-dependent increase in heart rate in response to noradrenaline administration in the physiological range to the vascular channel. Moreover, fluorescence live imaging of the intracellular calcium revealed synchronous fluorescence propagation at regular intervals in the myocardial channel and rhythmic excitation propagation without arrhythmia. Immunocytochemical analysis, in which the cardiac troponin T that constitutes the myocardial contraction machinery and the HCN4 channel expressed in pacemaker cells were stained, suggested that contracting cardiomyocytes and pacemaker cells constitute independent populations. Furthermore, when a pressure of 60 mmHg or a stretch stimulus of 1%, which simulates hypertension, was applied, nitric oxide release from vascular endothelial cells, which is expected in the actual heart, was confirmed by live imaging. This human heart-on-a-chip better reflects the functional characteristics of the heart and is expected to provide more accurate results in pharmacological and toxicological drug testing compared to laboratory animals.

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Event date： 2022.11.5 - 2022.11.6

Language：English   Presentation type：Oral presentation (general)  

Venue：Kochi  

Pulmonary fibrosis is a severe lung disease that causes scarring and thickening of the lungs, making it difficult for patients to breathe. Although animal models have often been used, models using human cells are necessary to develop better therapeutic methods for lung fibrosis. We first cultured a lung epithelial cell, NCI-H441, and administered transforming growth factor beta-1 to induce fibrosis-like changes to develop the human fibrosis model. Increased expression of fibrosis marker genes, including slug and vimentin in a dose-dependent manner, were revealed using quantitative reverse transcription polymerase chain reaction. Next, we used transwells to recapitulate the lung structure more faithfully. We seeded human umbilical vein endothelial cells on the basolateral side of the transwell coated with Matrigel, which facilitated cell adhesion. We then seeded cells NCI-H441 and fibroblasts on the apical side of the transwell. We removed the culture medium on the apical side to achieve an air-liquid interface (ALI) in the lung alveoli. The uptake of the intracellular calcium indicator was observed under a confocal microscope even two days later, suggesting successful ALI formation. Additionally, immunostaining of PECAM-1, a marker of the endothelial junction, indicated high integrity of the intercellular junction between endothelial cells. We will further develop this human triculture model to contribute to the development of treatments for pulmonary fibrosis.

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Event date： 2022.7.10 - 2022.7.14

Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：Taipei (hybrid)  

Mechanical stretch facilitates cardiomyocyte differentiation of human-induced pluripotent stem cells co-cultured with human gingival fibroblasts

Introduction
Although regenerative medicine using induced pluripotent stem cells (iPSCs) has been studied in cardiovascular medicine, scalability and efficiency of cardiomyocyte differentiation need to be improved. We hypothesized that mechanical stimulus could increase the efficiency and maturity of iPS-derived cardiomyocytes (iPS-CMs).

Methods
On a 2 × 2 cm elastic silicone chamber, we co-cultured human iPSCs with human gingival fibroblasts to promote differentiation of iPSCs into iPS-CMs and applied cyclic stretch (30 cycles/min) stimulus. We started a 72-h-long 2% stretch on the 15th day of cardiomyocyte differentiation protocol.

Results
Gene expression analysis using quantitative polymerase chain reaction revealed that expression levels of the mesoderm (Nkx2.5) and cardiomyocyte markers (cTnT) in the stretch group were significantly higher than those in the control group, suggesting that the stretch stimulus facilitated cardiomyocyte differentiation of iPSCs. Furthermore, expression levels of pluripotent markers (Sox2 and Oct4) tended to be lower in the stretch group than those in the control group. Furthermore, transmission electron microscopy revealed that the sarcomere length of iPS-CMs in the stretch group was significantly larger than that in the control group, suggesting that the stretch stimulus facilitated the maturation of iPS-CMs. Video-based contractility analysis suggested that the contractility of iPS-CMs tended to be larger in the stretch group than that in the control group.

Discussion
These results suggest that the mechanical stretch facilitates cardiomyocyte differentiation of iPSCs co-cultured with fibroblasts. The protocol of the stretch stimulus should be optimized in future studies.

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Event date： 2022.6.28 - 2022.6.30

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：Niigata  

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Event date： 2021.6.15 - 2021.6.17

Language：English   Presentation type：Oral presentation (general)  

Venue：online  

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Event date： 2020.5.25 - 2020.5.27

Language：English   Presentation type：Poster presentation  

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Event date： 2016.7.29 - 2016.7.30

Language：Japanese   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

Venue：Sendai (hybrid)  

The kidney is an important organ that controls the reabsorption and excretionof substances such as glucose, urea nitrogen, and creatinine. To develop amodel of human kidney, we used human renal proximal tubular epithelialcells (RPTECs) and human umbilical vein endothelial cells (HUVECs) torecapitulate the renal proximal tubule of the nephron. RPTECs and HUVECswere seeded on a two-channel microfluidic chip connected to a peristalticpump. To evaluate the barrier integrity of the cell layers, we measuredpermeability using Texas Red-conjugated dextran (MW: 3000). The apparentpermeability of the RPTEC-HUVEC bilayer was ~2 × 10
cm/s, suggestingsufficient barrier integrity. Next, we performed functional analysis of thekidney chip using urea nitrogen as an index. The concentration of ureanitrogen was ~0.7 mg/dl in the RPTEC channel, while it was ~0.3 mg/dl in theHUVEC channel, suggesting transport of urea nitrogen from the microvascularendothelial side to the kidney epithelial tubular side was active. However, theconcentration of glucose was ~110 mg/dl in the RPTEC channel, while it was~110 mg/dl in the HUVEC channel, suggesting no significant transport ofglucose. These results suggest that medium composition should be optimizedto assess the renal function of the kidney-on-a-chip.

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：Online  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：Online  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Oral presentation (keynote)  

Venue：Taipei  

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Okinawa  

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Language：English   Presentation type：Oral presentation (general)  

Venue：Takamatsu, Japan  

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Language：English   Presentation type：Poster presentation  

Venue：Takamatsu, Japan  

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Language：English   Presentation type：Oral presentation (general)  

Venue：Takamatsu, Japan  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：徳島  

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Language：English   Presentation type：Oral presentation (general)  

Venue：Barcelona, Spain  

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Language：English   Presentation type：Poster presentation  

Venue：Hamamatsu, Japan  

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Language：English   Presentation type：Poster presentation  

Venue：Hamamatsu, Japan  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：Sendai, Japan  

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Language：English   Presentation type：Poster presentation  

Venue：New Orleans, USA  

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Language：English   Presentation type：Poster presentation  

Venue：San Francisco  

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Language：English   Presentation type：Poster presentation  

Venue：San Francisco  

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Language：English   Presentation type：Poster presentation  

Venue：Singapore  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：旭川  

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Qingdao, China  

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Language：English   Presentation type：Poster presentation  

Venue：Milan, Italy  

Cellular scaffold is extremely important for cellular differentiation. To find efficient method to induce cardiac differentiation of stem cells, we tested several scaffold proteins using two dimensional (2D) and three dimensional (3D) cell culture systems. It is suggested that specific proteins such as vitronectin are necessary for 2D adhesion of stem cells and their cardiac differentiation. Also suggested is that fibroblast may play an essential role in forming 3D structure of cardiac tissue.

SaBPoT7.14

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Language：English   Presentation type：Poster presentation  

Venue：Okazaki, Japan  

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Language：English   Presentation type：Poster presentation  

Venue：Philadelphia, USA  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：岡山  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：Okayama, Japan  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：Okayama, Japan  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：Kagoshima, Japan  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：岡山  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岡崎  

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Suzhou, China  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：大宮  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：倉敷  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：Matsumoto, Japan  

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Beijing, China  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Dalian  

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Seoul  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：Aalborg  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：幕張  

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Yokohama  

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Nagoya  

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Language：English   Presentation type：Poster presentation  

Venue：San Diego, USA  

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Finite element analysis simulation of stress in human body

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てんかん・不整脈の創薬スクリーニングに向けた平面パッチクランプ装置の開発

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小型・安価な平面パッチクランプ装置

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Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

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Award type：Award from publisher, newspaper, foundation, etc.  Country：Japan

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Award type：Award from publisher, newspaper, foundation, etc.  Country：Japan

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Award type：Award from publisher, newspaper, foundation, etc.  Country：Japan

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Award type：Award from international society, conference, symposium, etc.  Country：Taiwan, Province of China

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Country：Japan

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Author：Other 

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