研究者詳細 - 清水　一憲

Discovery of fibroblast growth factor 2-derived peptides for enhancing mice skeletal muscle satellite cell proliferation

Quercetin induces a slow myofiber phenotype in engineered human skeletal muscle tissues Open Access

Nagai, A; Kaneda, Y; Izumo, T; Nakao, Y; Honda, H; Shimizu, K

FASEB JOURNAL 38 巻 ( 16 ) 頁： e70009 2024年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：FASEB Journal

Skeletal muscle comprises slow and fast myofibers, with slow myofibers excelling in aerobic metabolism and endurance. Quercetin, a polyphenol, is reported to induce slow myofibers in rodent skeletal muscle both in vitro and in vivo. However, its effect on human myofiber types remains unexplored. In this study, we evaluated quercetin's impact on slow myofiber induction using human skeletal muscle satellite cells. In a two-dimensional culture, quercetin enhanced gene expression, contributing to muscle differentiation, and significantly expanded the area of slow-type myosin heavy chain positive cells. It also elevated the gene expression of Pgc1α, an inducer of slow myofibers. Conversely, quercetin did not affect mitochondrial abundance, fission, or fusion, but it did increase the gene expression of Cox7A2L, which aids in promoting mitochondrial supercomplexity and endurance, and Mb, which contributes to oxidative phosphorylation. In a three-dimensional culture, quercetin significantly extended the time to peak tension and half relaxation time of the engineered human skeletal muscle tissues constructed on microdevices. Moreover, quercetin enhanced the muscle endurance of the tissues and curbed the rise in lactate secretion from the exercised tissues. These findings suggest that quercetin may induce slow myofibers in human skeletal muscle.

DOI： 10.1096/fj.202400914RR

Fujii, I; Kinoshita, R; Akiyama, H; Nakamura, A; Iwamori, K; Fukada, S; Honda, H; Shimizu, K

BIOTECHNOLOGY JOURNAL 19 巻 ( 8 ) 2024年8月

Bone morphogenetic protein signaling inhibitor improves differentiation and function of 3D muscle construct fabricated using C2C12

Ding, R; Xi, Y; Ito, A; Shimizu, K; Nagamori, E; Fujita, H; Kawamoto, T; Horie, M

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 137 巻 ( 6 ) 頁： 480 - 486 2024年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Bioscience and Bioengineering

Functional tissue-engineered artificial skeletal muscle tissue has great potential for pharmacological and academic applications. This study demonstrates an in vitro tissue engineering system to construct functional artificial skeletal muscle tissues using self-organization and signal inhibitors. To induce efficient self-organization, we optimized the substrate stiffness and extracellular matrix (ECM) coatings. We modified the tissue morphology to be ring-shaped under optimized self-organization conditions. A bone morphogenetic protein (BMP) inhibitor was added to improve overall myogenic differentiation. This supplementation enhanced the myogenic differentiation ratio and myotube hypertrophy in two-dimensional cell cultures. Finally, we found that myotube hypertrophy was enhanced by a combination of self-organization with ring-shaped tissue and a BMP inhibitor. BMP inhibitor treatment significantly improved myogenic marker expression and contractile force generation in the self-organized tissue. These observations indicated that this procedure may provide a novel and functional artificial skeletal muscle for pharmacological studies.

DOI： 10.1016/j.jbiosc.2024.03.005

Separation and enrichment of multiple bile acid micelle-disrupting peptides by adsorption/desorption process with heat-treated porous silica gels

Iriyama, M; Hagawa, H; Shimizu, S; Akiyama, H; Shimizu, K; Honda, H

BIOCHEMICAL ENGINEERING JOURNAL 205 巻 2024年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical Engineering Journal

Bile acid (BA) micelle-disrupting peptides are bioactive peptides (BPs) that suppress intestinal cholesterol adsorption and ameliorate blood cholesterol levels. To facilitate their application in the food industry, an effective and straightforward technology for BP separation and enrichment of BPs is required. For this purpose, we assessed the potential of heat-treated (HT) porous silica gels with an enhanced capacity for peptide adsorption/desorption (AD). Given the key characteristics of these peptides involving basic amino acid (AA) residues, we applied casein hydrolysate to HT silica gels for adsorption at neutral pH, where the surface yielded anionic silanolates through deprotonation. Subsequently, desorption was conducted at pH 2, at which point the surface returned to its neutral state. We confirmed a 335-fold increase in the BA micelle-disrupting activity of the resulting hydrolysates. LC-MS/MS analysis identified 783 peptides, confirming that the peptides containing cationic AA residues were selectively enriched, as expected. Moreover, a dose-response of randomly selected 27 peptides with various enrichment levels revealed the presence of multiple active peptides in the enriched groups. These results indicate that our method is effective for the separation and enrichment of BA micelle-disrupting peptides and holds promise for BP production.

DOI： 10.1016/j.bej.2024.109283

Electrical pulse stimulation-induced tetanic exercise simulation increases the secretion of extracellular vesicles from C2C12 myotubes

Murata, A; Akiyama, H; Honda, H; Shimizu, K

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 672 巻頁： 177 - 184 2023年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Extracellular vesicles (EVs) released into the blood during exercise mediate its whole-body health effects. The differentiation of EVs released by skeletal muscle cells in vivo from those released by other cells is challenging, therefore, it is unclear whether exercise increases the number of EVs secreted by skeletal muscle cells. In this study, we investigated whether exercise affects the quantity of EVs released from skeletal muscle cells using in vitro exercise models. C2C12 myotubes were cultured on a gel layer with 1 or 30 Hz electrical pulse stimulation (EPS) to induce contractions as an artificial simulating exercise. We found that tetanic contraction induced by 30 Hz EPS increased the number of secreted EVs. MicroRNA (miRNA)-seq analysis revealed that 30 Hz EPS altered the miRNA in the secreted EVs. Furthermore, expression analysis of genes related to the biogenesis and transport of EVs revealed that the expression of ALG-2 interacting protein X (Alix) was increased in response to 30 Hz EPS, and the peak value of intracellular Ca2+ in myotubes at 30 Hz EPS was higher than that at 1 Hz, indicating that the increase in intracellular Ca2+ concentration may be related to the increased secretion of EVs in response to 30 Hz EPS.

DOI： 10.1016/j.bbrc.2023.06.054

l-Anserine Increases Muscle Differentiation and Muscle Contractility in Human Skeletal Muscle Cells

Nagai, A; Ida, M; Izumo, T; Nakai, M; Honda, H; Shimizu, K

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 71 巻 ( 23 ) 頁： 8952 - 8958 2023年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Agricultural and Food Chemistry

l-Anserine, an imidazole peptide, has a variety of physiological activities, but its effects on skeletal muscle differentiation and muscle contractile force remain unknown. Thus, in this study, we investigated the effect of l-anserine on muscle differentiation and muscle contractile force in human skeletal muscle cells. In two-dimensional culture, 1 μM l-anserine significantly increased the myotube diameters (26.5 ± 1.71, 27.7 ± 1.08, and 28.8 ± 0.85 μm with 0, 0.1, and 1 μM l-anserine, respectively) and the expression levels of genes involved in muscle differentiation and the sarcomere structure. In three-dimensional culture, 1 μM l-anserine significantly increased the contractile force of engineered human skeletal muscle tissues cultured on a microdevice (1.99 ± 0.30, 2.17 ± 0.62, 2.66 ± 0.39, and 3.28 ± 0.85 μN with 0, 0.1, 0.5, and 1 μM l-anserine, respectively). l-Anserine also increased the myotube diameters and the proportion of myotubes with sarcomere structures in the cultured tissues. Furthermore, the histamine receptor 1 (H1R) antagonist attenuated the l-anserine-induced increase in the contractile force, suggesting the involvement of H1R in the mechanism of action of l-anserine. This study showed for the first time that l-anserine enhances muscle differentiation and muscle contractility via H1R.

DOI： 10.1021/acs.jafc.3c01685

Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling Open Access

Rashid, MI; Ito, T; Miya, F; Shimojo, D; Arimoto, K; Onodera, K; Okada, R; Nagashima, T; Yamamoto, K; Khatun, Z; Shimul, RI; Niwa, J; Katsuno, M; Sobue, G; Okano, H; Sakurai, H; Shimizu, K; Doyu, M; Okada, Y

SCIENTIFIC REPORTS 13 巻 ( 1 ) 頁： 8146 2023年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) differentiated into affected cell types can potentially recapitulate disease pathology more accurately than existing disease models. Such successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection, and clonal variations must be overcome. Moreover, their functionality should be carefully examined. Here, we demonstrated that bulk MYOD1-hiPSCs established with puromycin selection rather than G418 selection showed rapid and highly efficient differentiation. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties of clonally established MYOD1-hiPSCs, suggesting that it is possible to minimize clonal variations. Moreover, disease-specific hiPSCs of spinal bulbar muscular atrophy (SBMA) could be efficiently differentiated via this method into skeletal muscle that showed disease phenotypes, suggesting the applicability of this method for disease analysis. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical stimulation, indicating their functionality. Thus, our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and may facilitate the generation of muscular disease models.

DOI： 10.1038/s41598-023-34445-9

Drug-preloadable methacrylated gelatin microspheres fabricated using an aqueous two-phase system

Mizukami, Y; Yamaguchi, T; Shiono, M; Takahashi, Y; Shimizu, K; Konishi, S; Takakura, Y; Nishikawa, M

EUROPEAN POLYMER JOURNAL 181 巻 2022年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：European Polymer Journal

Gelatin microspheres (GMS) can retain biomolecules and be incorporated into artificial tissues and organs for tissue regeneration. However, the inactivation of biomolecules during the conventional fabrication process of GMS requires postloading of biomolecules to prefabricated GMS. Herein, a novel method for fabricating drug-preloaded GMS using a water-in-water (w/w) emulsification technique, is reported. A highly concentrated gelatin methacrylate (Gm) solution containing fibroblast growth factor 2 (FGF2), a model drug, was emulsified in a highly concentrated polyethylene glycol solution, and the mixture was irradiated with ultraviolet light. The obtained water-in-water gelatin methacrylate microspheres (w/w GmMS) were superior to conventional GMS in terms of stability, FGF2-loading efficiency, and sustained release of FGF2. The FGF2-loaded w/w GmMS efficiently activated the proliferation of adipose tissue-derived mesenchymal stem cells (ASCs). In addition, FGF2-loaded w/w GmMS incorporated into multicellular spheroids of ASCs increased the viability of ASCs in the spheroids. The therapeutic efficacy of ASC spheroids in a wound healing mouse model was significantly improved by the incorporation of FGF2-loaded w/w GmMS. These results indicate that drug-preloadable w/w GmMS can be widely applied in tissue engineering and regenerative medicine strategies.

DOI： 10.1016/j.eurpolymj.2022.111671

Quality evaluation of cell spheroids for transplantation by monitoring oxygen consumption using an on-chip electrochemical device. Open Access

Tsujimura M, Kusamori K, Takamura K, Ito T, Kaya T, Shimizu K, Konishi S, Nishikawa M

Biotechnology reports (Amsterdam, Netherlands) 36 巻頁： e00766 2022年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biotechnology Reports

Three-dimensional cell spheroids are superior cell-administration form for cell-based therapy which generally exhibit superior functionality and long-term survival after transplantation. Here, we nondestructively measured the oxygen consumption rate of cell spheroids using an on-chip electrochemical device (OECD) and examined whether this rate can be used as a marker to estimate the quality of cell spheroids. Cell spheroids containing NanoLuc luciferase-expressing mouse mesenchymal stem cell line C3H10T1/2 (C3H10T1/2/Nluc) were prepared. Spheroids of high or low quality were prepared by altering the medium change frequency. After transplantation into mice, the high-quality C3H10T1/2/Nluc spheroids exhibited a higher survival rate than the low-quality ones. The oxygen consumption rate of the high-quality C3H10T1/2/Nluc spheroids was maintained at high levels, whereas that of the low-quality spheroids decreased with time. These results indicate that OECD-based measurement of the oxygen consumption rate can be used to estimate the quality of cell spheroids without destructive analysis of the spheroids.

DOI： 10.1016/j.btre.2022.e00766

Alignment of Skeletal Muscle Cells Facilitates Acetylcholine Receptor Clustering and Neuromuscular Junction Formation with Co-Cultured Human iPSC-Derived Motor Neurons Open Access

Shimizu, K; Kassai, H; Kamei, Y; Yamamoto, K; Nagashima, T; Maekawa, T; Akiyama, H; Honda, H

CELLS 11 巻 ( 23 ) 2022年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cells

In vitro neuromuscular junction (NMJ) models are powerful tools for studying neuromuscular disorders. Although linearly patterned culture surfaces have been reported to be useful for the formation of in vitro NMJ models using mouse motor neuron (MNs) and skeletal muscle (SkM) myotubes, it is unclear how the linearly patterned culture surface increases acetylcholine receptor (AChR) clustering, one of the steps in the process of NMJ formation, and whether this increases the in vitro NMJ formation efficiency of co-cultured human MNs and SkM myotubes. In this study, we investigated the effects of a linearly patterned culture surface on AChR clustering in myotubes and examined the possible mechanism of the increase in AChR clustering using gene expression analysis, as well as the effects of the patterned surface on the efficiency of NMJ formation between co-cultured human SkM myotubes and human iPSC-derived MNs. Our results suggest that better differentiation of myotubes on the patterned surface, compared to the flat surface, induced gene expression of integrin α7 and AChR ε-subunit, thereby increasing AChR clustering. Furthermore, we found that the number of NMJs between human SkM cells and MNs increased upon co-culture on the linearly patterned surface, suggesting the usefulness of the patterned surface for creating in vitro human NMJ models.

DOI： 10.3390/cells11233760

Analysis of plasmodesmata permeability using cultured tobacco BY-2 cells entrapped in microfluidic chips

Kurotani, K; Kawakatsu, Y; Kikkawa, M; Tabata, R; Kurihara, D; Honda, H; Shimizu, K; Notaguchi, M

JOURNAL OF PLANT RESEARCH 135 巻 ( 5 ) 頁： 693 - 701 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Plant Research

Plasmodesmata are unique channel structures in plants that link the fluid cytoplasm between adjacent cells. Plants have evolved these microchannels to allow trafficking of nutritious substances as well as regulatory factors for intercellular communication. However, tracking the behavior of plasmodesmata in real time is difficult because they are located inside tissues. Hence, a system was constructed to monitor the movement of substances by plasmodesmata using tobacco BY-2 cells, which are linearly organized cells, and a microfluidic device that traps them in place and facilitates observation. After targeting one cell for photobleaching, recovery of the lost H2B-GFP protein was detected within 200 min. No recovery was detected in that time frame by photobleaching the entire cell filaments. This suggested that the recovery of H2B-GFP protein was not due to de novo protein synthesis, but rather to translocation from neighboring cells. The transport of H2B-GFP protein was not observed when sodium chloride, a compound known to cause plasmodesmata closure, was present in the microfluid channel. Thus, using the microfluidic device and BY-2 cells, it was confirmed that the behavior of plasmodesmata could be observed in real time under controllable conditions.

DOI： 10.1007/s10265-022-01406-8

Screening of anti-atrophic peptides by using photo-cleavable peptide array and 96-well scale contractile human skeletal muscle atrophy models

Yamamoto, K; Ohsumi, S; Nagashima, T; Akiyama, H; Honda, H; Shimizu, K

BIOTECHNOLOGY AND BIOENGINEERING 119 巻 ( 8 ) 頁： 2196 - 2205 2022年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biotechnology and Bioengineering

Skeletal muscle atrophy is characterized by decreases in protein content, myofiber diameter, and contractile force generation. As muscle atrophy worsens the quality of life, the development of anti-atrophic substances is desirable. In this study, we aimed to demonstrate a screening process for anti-atrophic peptides using photo-cleavable peptide array technology and human contractile atrophic muscle models. We developed a 96-well system and established a screening process with less variability. Dexamethasone-induced human atrophic tissue was constructed in the system. Eight peptides were selected from the literature and used for the screening of peptides for preventing the decrease of the contractile forces of tissues. The peptide QIGFIW, which showed preventive activity, was selected as the seed sequence. As a result of amino acid substitution, we obtained QIGFIQ as a peptide with higher anti-atrophic activity. These results indicate that the combinatorial use of the photo-cleavable peptide array technology and 96-well screening system could comprise a powerful approach to obtaining anti-atrophic peptides, and suggest that the 96-well screening system and atrophic model represent a practical and powerful tool for the development of drugs/functional food ingredients.

DOI： 10.1002/bit.28125

Selective concentration of antimicrobial peptides to heat-treated porous silica gel using adsorption/desorption Open Access

Mechanical Regulation Of Provisional Matrix Assembly In 3D Fibrous Microtissues 国際共著

Eyckmans, J; Das, S; Shimizu, K; Chen, C

WOUND REPAIR AND REGENERATION 30 巻 ( 2 ) 頁： A20 - A21 2022年3月

Hagawa, H; Imai, K; Gao, ZW; Taniguchi, M; Shimizu, K; Honda, H

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 133 巻 ( 2 ) 頁： 161 - 167 2022年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Bioscience and Bioengineering

Heat-treated porous silica gel (HT silica gel) previously developed by our group has selectively adsorbed cationic peptides at a pH of 7. Therefore, we focused on the use of antimicrobial peptides (AMPs) as bioactive peptides (BPs). First, 32 AMPs and 32 randomly designed peptides were generated using Fmoc solid synthesis, and their adsorption ratio to HT-silica gel was investigated. Thirty two AMPs showed a relatively higher adsorption ratio of 58.8% compared to that of randomly designed peptides, which was 35.3%. Desorption conditions were investigated using Amyl-1-18 antimicrobial peptides. Next, pepsin hydrolysate from rice endosperm protein (REP) powder was prepared by ourselves. The REP hydrolysate containing dry matter (7.5 mg) was applied to the adsorption/desorption (AD) procedure using HT silica gel to obtain 1.6 mg of AD hydrolysate. When the two hydrolysates were subjected to mass spectrometry, 305 concentrated peptides were obtained. In total, 26 peptides with high content and high enrichment ratios were listed and synthesized. When the antimicrobial activity of these 26 peptides was evaluated using Cutibacterium acnes, five peptides consisting of 12–27 amino acids were identified as novel AMPs. Two of these peptides, which were derived from rice glutelin, showed antimicrobial activity against all four microbes, including Porphyromonas gingivalis, Escherichia coli, and Streptococcus mutans. In the present study, we showed that AMPs could be easily enriched from protein hydrolysate using HT silica gel. The adsorption/desorption procedure using HT silica gel was confirmed to be a useful tool for convenient BP separation.

DOI： 10.1016/j.jbiosc.2021.11.002

Intravenous injection of mesenchymal stem cell spheroids improves the pulmonary delivery and prolongs in vivo survival

Shimazawa, Y; Kusamori, K; Tsujimura, M; Shimomura, A; Takasaki, R; Takayama, Y; Shimizu, K; Konishi, S; Nishikawa, M

BIOTECHNOLOGY JOURNAL 17 巻 ( 1 ) 頁： e2100137 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biotechnology Journal

Background: Because of the excellent therapeutic potential, mesenchymal stem cells (MSCs) have been used as cell therapeutics for various diseases. However, the survival rate and duration of MSCs after transplantation are extremely low and short, respectively. To solve these problems, in this study, we prepared multicellular spheroids of MSCs and investigated their survival and function after intravenous injection in mice. Methods and Results: The murine adipose-derived MSC line m17.ASC was cultured in agarose-based microwell plates to obtain size-controlled m17.ASC spheroids of an average diameter and cell number of approximately 170 μm and 1100 cells/spheroid, respectively. The intravenously injected m17.ASC spheroids mainly accumulated in the lung and showed a higher survival rate than suspended m17.ASC cells during the experimental period of 7 days. m17.ASC spheroids efficiently reduced the lipopolysaccharide-induced increase in plasma concentrations of interleukin-6 and tumor necrosis factor-α. Conclusions: These results indicate that spheroid formation improved the pulmonary delivery and survival of MSCs, as well as their therapeutic potential against inflammatory pulmonary diseases.

DOI： 10.1002/biot.202100137

Detection of protease digestion site using fluorescence labeled peptide array and construction of machine-learning prediction model

Development of microfluidic chip for entrapping tobacco BY-2 cells Open Access

Shimizu, K; Kawakatsu, Y; Kurotani, KI; Kikkawa, M; Tabata, R; Kurihara, D; Honda, H; Notaguchi, M

PLOS ONE 17 巻 ( 4 ) 頁： e0266982 2022年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PLoS ONE

The tobacco BY-2 cell line has been used widely as a model system in plant cell biology. BY-2 cells are nearly transparent, which facilitates cell imaging using fluorescent markers. As cultured cells are drifted in the medium, therefore, it was difficult to observe them for a long period. Hence, we developed a microfluidic device that traps BY-2 cells and fixes their positions to allow monitoring the physiological activity of cells. The device contains 112 trap zones, with parallel slots connected in series at three levels in the flow channel. BY-2 cells were cultured for 7 days and filtered using a sieve and a cell strainer before use to isolate short cell filaments consisting of only a few cells. The isolated cells were introduced into the flow channel, resulting in entrapment of cell filaments at 25 out of 112 trap zones (22.3%). The cell numbers increased through cell division from 1 to 4 days after trapping with a peak of mitotic index on day 2. Recovery experiments of fluorescent proteins after photobleaching confirmed cell survival and permeability of plasmodesmata. Thus, this microfluidic device and one-dimensional plant cell samples allowed us to observe cell activity in real time under controllable conditions.

DOI： 10.1371/journal.pone.0266982

Mizutani R., Mori Y., Ogawa S., Tazoe K., Akiyama H., Shimizu K., Honda H.

Seibutsu-kogaku Kaishi 100 巻 ( 10 ) 頁： 528 - 540 2022年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Seibutsu-kogaku Kaishi

For the prediction of protease digestion site, 1990 kinds of tetramer peptide were designed as a library, of which N terminal end was fluorescence labelled. Decrease of fluorescence intensity of each peptide was quantitatively determined and used as a training data for Random Forest (RF) modeling. All of dipeptides bond as a protease substrate were included in 1990 tetramer peptides. As an explanatory variable for model construction, 532 parameters were prepared and those were included not only appearance of amino acid residue or dipeptides but also positioning parameters (PP) or global parameters (GP) to explain the peptide property. Trypsin for biochemistry grade was used for hydrolysis. After digesting at pH 8, the histogram of digestion ratio was appeared that tetramers with arginine residue (R) and lysine residue (K) could be expectedly digested at higher ratio compared with tetramers without R or K. Constructed pH 8-RF model showed 77 % of prediction accuracy. The explanatory parameters with top 10 higher importance in pH 8-RF model were both of appearance of K and 9 GPs. GPs were including 4 isoelectric parameters and 1 polarity parameter, of which K or R residue have relatively large value. Digestion site of α-lactalbumin was determined using pH 8-RF model and the protein was actually digested by trypsin. When the hydrolysate was analyzed by LC-MS/MS, 42 peptides were identified. Fourteen sites among 19 predicted digestion sites were coincided with each other. Many peptide fragments digested at 69Y-70G site or 50F-51H site was detected and it was strongly suggested to be digested by chymotrypsin as a contaminated protease. These sites were fairly predicted by constructed RF model. In addition, trypsin digestion at pH 5 was carried out to investigate the effect of pH decrease on trypsin digestion. The prediction accuracy of pH 5-RF model was only 59 %. The parameters with top 10 higher importance in pH 5-RF model were including 4 GPs which was the same with 4 isoelectric parameter in pH 8 model. Twenty digestion sites of α-lactalbumin were predicted from pH 5-RF model. α-lactalbumin was digested at pH 5 by trypsin, and 64 peptides were identified. The predicted sites were compared with identified fragments at the region from 48T to 77K in which a lot of fragments were detected. It was concluded that constructed model could fairly predict some of newly identified sites and could roughly grasp slight modification of digestion site by pH change. The proposed methodology, which contained 1990 kinds of tetramer peptides as a library, 532 explanatory parameters and RF model, was effective for prediction of protease digestion site.

DOI： 10.34565/seibutsukogaku.100.10_528

Agonist/Antagonist Activity of Oxytocin Variants Obtained from Free Cyclic Peptide Libraries Generated via Amino Acid Substitution Open Access

Kinoshita, R; Kozaki, I; Shimizu, K; Shibata, T; Ochiai, A; Honda, H

ACS OMEGA 6 巻 ( 46 ) 頁： 31244 - 31252 2021年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACS Omega

We established a method for synthesizing a free cyclic peptide library via peptide array synthesis to demonstrate the sequence activity of cyclic peptides. Variants of the cyclic nonapeptide oxytocin (OXT) were synthesized via residue substitution. Natural amino acids (AAs) were classified into eight groups based on their physical properties and the size of their side chains, and a representative AA from each group was selected for residue substitution. All OXT variants were systematically evaluated for agonist/antagonist activity. Consequently, no improvement in agonist activity was observed, although substitution of the P4 and P8 residues resulted in decreased activity due to AA substitution. A few OXT variants exhibited antagonistic activity. In particular, the variants with P2 Leu residue substitution (Y2L) and Phe substitutions at residues 4 (Q4F), 5 (N5F), and 7 (P7F) showed high antagonistic activity. Variant Y2W was found to have the highest inhibitory effect, with a dissociation constant of 44 nM, which was comparable to that of the commercial antagonist atosiban (21 nM). Therefore, a free cyclic peptide library constructed via substitution with a natural AA residue was confirmed to be a powerful tool for bioactive peptide screening.

DOI： 10.1021/acsomega.1c04982

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply Open Access

Mizukami, Y; Takahashi, Y; Shimizu, K; Konishi, S; Takakura, Y; Nishikawa, M

BIOLOGICAL & PHARMACEUTICAL BULLETIN 44 巻 ( 10 ) 頁： 1458 - 1464 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biological and Pharmaceutical Bulletin

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16–BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

DOI： 10.1248/bpb.b21-00269

Microarray profiling of gene expression in C2C12 myotubes trained by electric pulse stimulation

Fujita, H; Horie, M; Shimizu, K; Nagamori, E

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 132 巻 ( 4 ) 頁： 417 - 422 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Bioscience and Bioengineering

Electric pulse-stimulated C2C12 myotubes are gaining interest in the field of muscle physiology and biotechnology because electric pulse stimulation (EPS) enhances sarcomere structure development and active tension generation capability. Recently, we found that termination of EPS results in the rapid loss of active tension generation accompanied by disassembly of the sarcomere structure, which may represent an in vitro muscle atrophy model. To elucidate the molecular mechanism underlying this rapid loss of active tension generation and sarcomere structure disassembly after termination of EPS, we performed transcriptomic analysis using microarray. After termination of EPS, 74 genes were upregulated and 120 genes were downregulated after 30 min; however, atrophy-related genes were not found among these genes. To further assess the effect of EPS on gene expression, we re-applied EPS after its termination for 8 h and searched for genes whose expression was reversed. Four genes were upregulated by termination of EPS and downregulated by the re-application of EPS, whereas two genes were downregulated by termination of EPS and upregulated by the re-application of EPS. Although none of these genes were atrophy- or hypertrophy-related, the results presented in this study will contribute to the understanding of gene expression changes that mediate rapid loss of active tension generation and sarcomere structure disassembly following termination of EPS in C2C12 myotubes.

DOI： 10.1016/j.jbiosc.2021.06.016

In Silico Screening of a Bile Acid Micelle Disruption Peptide for Oral Consumptions from Edible Peptide Database Open Access

Imai, K; Takeuchi, Y; Shimizu, K; Honda, H

FOODS 10 巻 ( 10 ) 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Foods

Recently, many bioactive peptides have been identified using bioinformatics tools. Previously, our group developed a method to screen dual-functional peptides that have direct intestinal delivery with porous silica gel and bile acid micelle disruption. However, newly designed peptides were not found in any storage protein. Therefore, in this study, in silico screening was performed using a 350,000 edible peptide library consisting of 4-to 7-mer independent peptides. As an initial screening, all edible peptides were applied to the random forest model to select predicted positive peptides. For a second screening, the peptides were assessed for the possibility of intestinal delivery using a 3D color map. From this approach, three novel dual-functional peptides, VYVFDE, WEFIDF, and VEEFYC were identified, and all of them were derived from storage proteins (legumin, myosin, and 11S globulin). In particular, VEEFYCS, in which a serine residue (S) is added to VEEFYC, was assumed to be released by thermolysin from the 11S-globulin derived from Ginkgo biloba by LC-MS/MS analysis. VEEFYCS was found to have suitable direct intestinal delivery and bile acid micelle disruption activity.

DOI： 10.3390/foods10102496

Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions

Ishihara, M; Kikkawa, M; Shimizu, K; Suzuki, H; Honda, H

BIOTECHNOLOGY LETTERS 43 巻 ( 9 ) 頁： 1905 - 1911 2021年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biotechnology Letters

Objectives: To develop a simple pectin-degrading microorganism screening method. Results: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. Conclusions: The developed method is useful for identifying pectin-degrading microorganisms.

DOI： 10.1007/s10529-021-03162-6

Machine learning screening of bile acid-binding peptides in a peptide database derived from food proteins Open Access

Imai, K; Shimizu, K; Honda, H

SCIENTIFIC REPORTS 11 巻 ( 1 ) 頁： 16123 2021年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Bioactive peptides (BPs) are protein fragments that exhibit a wide variety of physicochemical properties, such as basic, acidic, hydrophobic, and hydrophilic properties; thus, they have the potential to interact with a variety of biomolecules, whereas neither carbohydrates nor fatty acids have such diverse properties. Therefore, BP is considered to be a new generation of biologically active regulators. Recently, some BPs that have shown positive benefits in humans have been screened from edible proteins. In the present study, a new BP screening method was developed using BIOPEP-UWM and machine learning. Training data were initially obtained using high-throughput techniques, and positive and negative datasets were generated. The predictive model was generated by calculating the explanatory variables of the peptides. To understand both site-specific and global characteristics, amino acid features (for site-specific characteristics) and peptide global features (for global characteristics) were generated. The constructed models were applied to the peptide database generated using BIOPEP-UWM, and bioactivity was predicted to explore candidate bile acid-binding peptides. Using this strategy, seven novel bile acid-binding peptides (VFWM, QRIFW, RVWVQ, LIRYTK, NGDEPL, PTFTRKL, and KISQRYQ) were identified. Our novel screening method can be easily applied to industrial applications using whole edible proteins. The proposed approach would be useful for identifying bile acid-binding peptides, as well as other BPs, as long as a large amount of training data can be obtained.

DOI： 10.1038/s41598-021-95461-1

Development of a human neuromuscular tissue-on-a-chip model on a 24-well-plate-format compartmentalized microfluidic device 国際誌 Open Access

Yamamoto, K; Yamaoka, N; Imaizumi, Y; Nagashima, T; Furutani, T; Ito, T; Okada, Y; Honda, H; Shimizu, K

LAB ON A CHIP 21 巻 ( 10 ) 頁： 1897 - 1907 2021年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Lab on a Chip

Engineered three-dimensional models of neuromuscular tissues are promising for use in mimicking their disorder states in vitro. Although several models have been developed, it is still challenging to mimic the physically separated structures of motor neurons (MNs) and skeletal muscle (SkM) fibers in the motor units in vivo. In this study, we aimed to develop microdevices for precisely compartmentalized coculturing of MNs and engineered SkM tissues. The developed microdevices, which fit a well of 24 well plates, had a chamber for MNs and chamber for SkM tissues. The two chambers were connected by microtunnels for axons, permissive to axons but not to cell bodies. Human iPSC (hiPSC)-derived MN spheroids in one chamber elongated their axons into microtunnels, which reached the tissue-engineered human SkM in the SkM chamber, and formed functional neuromuscular junctions with the muscle fibers. The cocultured SkM tissues with MNs on the device contracted spontaneously in response to spontaneous firing of MNs. The addition of a neurotransmitter, glutamate, into the MN chamber induced contraction of the cocultured SkM tissues. Selective addition of tetrodotoxin or vecuronium bromide into either chamber induced SkM tissue relaxation, which could be explained by the inhibitory mechanisms. We also demonstrated the application of chemical or mechanical stimuli to the middle of the axons of cocultured tissues on the device. Thus, compartmentalized neuromuscular tissue models fabricated on the device could be used for phenotypic screening to evaluate the cellular type specific efficacy of drug candidates and would be a useful tool in fundamental research and drug development for neuromuscular disorders.

DOI： 10.1039/d1lc00048a

Electrospray propelled by ionic wind in a bipolar system for direct delivery of charge reduced nanoparticles 国際共著 Open Access

Dau, VT; Vu, TH; Tran, CD; Nguyen, TV; Nguyen, TK; Dinh, T; Phan, HP; Shimizu, K; Nguyen, NT; Dao, DV

APPLIED PHYSICS EXPRESS 14 巻 ( 5 ) 2021年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Applied Physics Express

We present a conceptual design to generate and deliver nanoparticles in one unique system based on electrohydrodynamic atomisation (EHDA) without the restriction of the collector. The present EHDA bipolar configuration consists of a capillary nozzle and a pin, both act as emitters and as the reference electrodes of each other. Under an applied voltage, the capillary nozzle sprays droplets while the pin generates ion wind via corona discharge. During spraying process, droplets' charge is significantly reduced by interacting with counter ions and propelled away from the electrodes by the momentum of ion winds accumulated from corona discharge. Thus, the present technique can yield promising applications in effective respiratory delivery of nanomedicine. © 2021 The Japan Society of Applied Physics.

DOI： 10.35848/1882-0786/abf36b

Screening of a novel free fatty acid receptor 1 (FFAR1) agonist peptide by phage display and machine learning based-amino acid substitution 査読有り

Yoshioka K.

Biochemical and Biophysical Research Communications 550 巻頁： 177 - 183 2021年4月

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character 査読有り

Kozaki I.

Journal of Peptide Science 27 巻 ( 1 ) 2021年1月

Screening of FFAR1-Activating Peptides by Molecular Structural Analysis

Yoshioka, K; Yamashita, H; Fujitani, M; Kato, R; Shimizu, K; Honda, H

KAGAKU KOGAKU RONBUNSHU 47 巻 ( 3 ) 頁： 64 - 68 2021年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Kagaku Kogaku Ronbunshu

To find FFAR1-activating peptides consisting of natural amino acid residues, molecular structure analysis was performed by hierarchical clustering using extraction of physicochemical parameters. When 3 mer to 6 mer peptide libraries were prepared and the molecular structure of each was compared with 5 chemical agonists, 12 peptides with high correlation coefficients of physicochemical parameters were found from the 6 mer peptide library. these peptides were then synthesized by Fmoc solid phase synthesis, and their activity was assessed by TGFα shedding assay using FFAR1-expressing HEK293. As a result, two peptides, GCGGSS and GASGCC, were identified as peptide agonists with relatively high activity. Although these peptides showed approximately one fithh of the activity of chemical agonist GW9508, they are the first examples that we know of FFAR1-activating peptides consisting of natural amino acid residues.

DOI： 10.1252/kakoronbunshu.47.64

Study on functional expression and evaluation of cultured skeletal muscle cells using microfabricated devices

Shimizu K

Seibutsu-kogaku Kaishi 99 巻 ( 3 ) 頁： 122 - 128 2021年

Machine Learning-Based Amino Acid Substitution of Short Peptides: Acquisition of Peptides with Enhanced Inhibitory Activities against α-Amylase and α-Glucosidase

Yamashita H.

ACS Biomaterials Science and Engineering 6 巻 ( 11 ) 頁： 6117 - 6125 2020年11月

Bile acid micelle disruption activity of short-chain peptides from tryptic hydrolyzate of edible proteins

Ito M.

Journal of Bioscience and Bioengineering 130 巻 ( 5 ) 頁： 514 - 519 2020年11月

In Vitro Model of Human Skeletal Muscle Tissues with Contractility Fabricated by Immortalized Human Myogenic Cells

Nagashima T.

Advanced Biosystems 4 巻 ( 11 ) 2020年11月

Effect of cell-extracellular matrix interaction on myogenic characteristics and artificial skeletal muscle tissue.

Ding R, Horie M, Nagasaka S, Ohsumi S, Shimizu K, Honda H, Nagamori E, Fujita H, Kawamoto T

Journal of bioscience and bioengineering 130 巻 ( 1 ) 頁： 98-105 - 105 2020年7月

Efficient capturing of circulating tumor cells using a magnetic capture column and a size-selective filter

Yamamoto S

Bioprocess and Biosystems Engineering 38 巻 ( 9 ) 2020年5月

Fabrication of contractile skeletal muscle tissues using directly converted myoblasts from human fibroblasts.

Shimizu K, Ohsumi S, Kishida T, Mazda O, Honda H

Journal of bioscience and bioengineering 129 巻 ( 5 ) 頁： 632-637 - 637 2020年5月

Disulfide linked hetero dimeric peptide arrays for screening functional peptides inside cells.

Kozaki I, Shimizu K, Honda H

Journal of bioscience and bioengineering 129 巻 ( 5 ) 頁： 613-618 - 618 2020年5月

Tissue suction-mediated gene transfer to the beating heart in mice

Taniguchi Yota, Oyama Natsuko, Fumoto Shintaro, Kinoshita Hideyuki, Yamashita Fumiyoshi, Shimizu Kazunori, Hashida Mitsuru, Kawakami Shigeru

PLOS ONE 15 巻 ( 2 ) 頁： e0228203 2020年2月

Miniaturized skeletal muscle tissue fabrication for measuring contractile activity

Yoshioka K.

Journal of Bioscience and Bioengineering 2020年

Incorporation of gelatin microspheres into hepg2 human hepatocyte spheroids for functional improvement through improved oxygen supply to spheroid core

Mizukami Y.

Biological and Pharmaceutical Bulletin 43 巻 ( 8 ) 頁： 1220 - 1225 2020年

Open-Chamber Co-Culture Microdevices for Single-Cell Analysis of Skeletal Muscle Myotubes and Motor Neurons with Neuromuscular Junctions

Yamaoka Nao, Shimizu Kazunori, Imaizumi Yu, Ito Takuji, Okada Yohei, Honda Hiroyuki

BIOCHIP JOURNAL 13 巻 ( 2 ) 頁： 127-132 2019年6月

Regulation of the Distribution of Cells in Mixed Spheroids by Altering Migration Direction

Mizukami Yuya, Takahashi Yuki, Shimizu Kazunori, Konishi Satoshi, Takakura Yoshinobu, Nishikawa Maikiya

TISSUE ENGINEERING PART A 25 巻 ( 5-6 ) 頁： 390-398 2019年3月

Searching for high-binding peptides to bile acid for inhibition of intestinal cholesterol absorption using principal component analysis.

Ito M, Shimizu K, Honda H

Journal of bioscience and bioengineering 127 巻 ( 3 ) 頁： 366-371 2019年3月

Regulation of the Distribution of Cells in Mixed Spheroids by Altering Migration Direction.

Mizukami Y, Takahashi Y, Shimizu K, Konishi S, Takakura Y, Nishikawa M

Tissue engineering. Part A 25 巻 ( 5-6 ) 頁： 390-398 2019年3月

Searching for high-binding peptides to bile acid for inhibition of intestinal cholesterol absorption using principal component analysis

Ito Masako, Shimizu Kazunori, Honda Hiroyuki

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 127 巻 ( 3 ) 頁： 366-371 2019年3月

Selective Elimination of Bitter Peptides by Adsorption to Heat-treated Porous Silica Gel 査読有り

Imai, K, Ikeda, A, Shimizu, K, Honda, H

Food Science and Technology Research 25 巻 ( 2 ) 頁： 179-186 - 186 2019年

Pep-MS assay: Protease hydrolysis assay system using photo-cleavable peptide array and mass spectrometer 査読有り

Kurimoto, M, Shimizu, K, Ochi, H, Abe, F, Honda, H

Journal of Bioscience and Bioengineering 128 巻 ( 2 ) 頁： 156-161 - 161 2019年

Predictive selection and evaluation of appropriate functional peptides for intestinal delivery with a porous silica gel 査読有り

Imai, K, Shimizu, K, Honda, H

Journal of Bioscience and Bioengineering 128 巻 ( 1 ) 頁： 44-49 - 49 2019年

Pep-MS assay: Protease hydrolysis assay system using photo-cleavable peptide array and mass spectrometer 査読有り

Kurimoto, M., Shimizu, K., Ochi, H., Abe, F., Honda, H.

Journal of Bioscience and Bioengineering 128 巻 ( 2 ) 頁： 156-161 2019年

Predictive selection and evaluation of appropriate functional peptides for intestinal delivery with a porous silica gel 査読有り

Imai, K., Shimizu, K., Honda, H.

Journal of Bioscience and Bioengineering 128 巻 ( 1 ) 頁： 44-49 2019年

Selective Elimination of Bitter Peptides by Adsorption to Heat-treated Porous Silica Gel 査読有り

Imai, K., Ikeda, A., Shimizu, K., Honda, H.

Food Science and Technology Research 25 巻 ( 2 ) 頁： 179-186 2019年

In-process evaluation of culture errors using morphology-based image analysis

Imai Yuta, Yoshida Kei, Matsumoto Megumi, Okada Mai, Kanie Kei, Shimizu Kazunori, Honda Hiroyuki, Kato Ryuji

REGENERATIVE THERAPY 9 巻頁： 15-23 2018年12月

Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine

Nagashima Takunori, Shimizu Kazunori, Matsumoto Ryo, Honda Hiroyuki

SCIENTIFIC REPORTS 8 巻 2018年8月

Interaction between porous silica gel microcarriers and peptides for oral administration of functional peptides

Imai Kento, Shimizu Kazunori, Kamimura Mitsuhiro, Honda Hiroyuki

SCIENTIFIC REPORTS 8 巻 2018年7月

Determining Transgene Expression Characteristics Using a Suction Device with Multiple Hole Adjusting a Left Lateral Lobe of the Mouse Liver

Haraguchi Ayana, Fuchigami Yuki, Kawaguchi Maho, Fumoto Shintaro, Ohyama Kaname, Shimizu Kazunori, Hagimori Masayori, Kawakami Shigeru

BIOLOGICAL & PHARMACEUTICAL BULLETIN 41 巻 ( 6 ) 頁： 944-950 2018年6月

Determining Transgene Expression Characteristics Using a Suction Device with Multiple Hole Adjusting a Left Lateral Lobe of the Mouse Liver

Haraguchi Ayana, Fuchigami Yuki, Kawaguchi Maho, Fumoto Shintaro, Ohyama Kaname, Shimizu Kazunori, Hagimori Masayori, Kawakami Shigeru

BIOLOGICAL & PHARMACEUTICAL BULLETIN 41 巻 ( 6 ) 頁： 944-950 2018年6月

Mutations responsible for alcohol tolerance in the mutant of Synechococcus elongatus PCC 7942 (SY1043) obtained by single-cell screening system

Hirokawa Yasutaka, Kanesaki Yu, Arai Sayuri, Saruta Fumiko, Hayashihara Kayoko, Murakami Akio, Shimizu Kazunori, Honda Hiroyuki, Yoshikawa Hirofumi, Hanai Taizo

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 125 巻 ( 5 ) 頁： 572-577 2018年5月

Control of polarization and tumoricidal activity of macrophages by multicellular spheroid formation

Tanaka Yutaro, Nishikawa Makiya, Mizukami Yuya, Kusamori Kosuke, Ogino Yuka, Nishimura Shunsuke, Shimizu Kazunori, Konishi Satoshi, Takahashi Yuki, Takakura Yoshinobu

JOURNAL OF CONTROLLED RELEASE 270 巻頁： 177-183 2018年1月

Determining Transgene Expression Characteristics Using a Suction Device with Multiple Hole Adjusting a Left Lateral Lobe of the Mouse Liver.

Haraguchi A, Fuchigami Y, Kawaguchi M, Fumoto S, Ohyama K, Shimizu K, Hagimori M, Kawakami S

Biological & pharmaceutical bulletin 41 巻 ( 6 ) 頁： 944 - 950 2018年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biological and Pharmaceutical Bulletin

We developed a tissue suction-mediated transfection method (suction method) as a relatively reliable and less invasive technique for in vivo transfection. In this study, we determined hepatic transgene expression characteristics in the mouse liver, using a suction device, collecting information relevant to gene therapy and gene functional analysis by the liver suction method. To achieve high transgene expression levels, we developed a suction device with four holes (multiple hole device) and applied it to the larger portion of the left lateral lobe of the mouse liver. Hepatic transfection with physical stimuli was potentially controlled by activator protein-1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We examined the spatial distribution of transgene expression in the suctioned lobe by 2-dimensional imaging with histo-chemical staining and 3-dimensional multicolor deep imaging with tissue clearing methods. Through monitoring spatial distribution of transgene expression, the liver suction method was used to efficiently transfect extravascular hepatocytes in the suction-deformable upper lobe of the liver. Moreover, long-term transgene expression, at least 14d, was achieved with the liver suction method when cytosine-phosphate-guanine (CpG)-free plasmid DNA was applied.

DOI： 10.1248/bpb.b18-00094

Characterization of transgene expression and pDNA distribution of the suctioned kidney in mice.

Oyama N, Fuchigami Y, Fumoto S, Sato M, Hagimori M, Shimizu K, Kawakami S

Drug delivery 24 巻 ( 1 ) 頁： 906-917 2017年11月

Morphology-based non-invasive quantitative prediction of the differentiation status of neural stem cells.

Fujitani M, Huddin NS, Kawai S, Kanie K, Kiyota Y, Shimizu K, Honda H, Kato R

Journal of bioscience and bioengineering 124 巻 ( 3 ) 頁： 351-358 2017年9月

Using size-controlled multicellular spheroids of murine adenocarcinoma cells to efficiently establish pulmonary tumors in mice.

Nishikawa T, Tanaka Y, Kusamori K, Mizuno N, Mizukami Y, Ogino Y, Shimizu K, Konishi S, Takahashi Y, Takakura Y, Nishikawa M

Biotechnology journal 12 巻 ( 8 ) 2017年8月

Effective modification of cell death-inducing intracellular peptides by means of a photo-cleavable peptide array-based screening system.

Kozaki I, Shimizu K, Honda H

Journal of bioscience and bioengineering 124 巻 ( 2 ) 頁： 209-214 2017年8月

Alcohol-tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single-cell mutant screening system

Arai Sayuri, Hayashihara Kayoko, Kanamoto Yuki, Shimizu Kazunori, Hirokawa Yasutaka, Hanai Taizo, Murakami Akio, Honda Hiroyuki

BIOTECHNOLOGY AND BIOENGINEERING 114 巻 ( 8 ) 頁： 1771-1778 2017年8月

Three-Dimensional Culture Model of Skeletal Muscle Tissue with Atrophy Induced by Dexamethasone. Open Access

Shimizu K, Genma R, Gotou Y, Nagasaka S, Honda H

Bioengineering (Basel, Switzerland) 4 巻 ( 2 ) 頁： 56 2017年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Bioengineering

Drug screening systems for muscle atrophy based on the contractile force of cultured skeletal muscle tissues are required for the development of preventive or therapeutic drugs for atrophy. This study aims to develop a muscle atrophy model by inducing atrophy in normal muscle tissues constructed on microdevices capable of measuring the contractile force and to verify if this model is suitable for drug screening using the contractile force as an index. Tissue engineered skeletal muscles containing striated myotubes were prepared on the microdevices for the study. The addition of 100 µM dexamethasone (Dex), which is used as a muscle atrophy inducer, for 24 h reduced the contractile force significantly. An increase in the expression of Atrogin-1 and MuRF-1 in the tissues treated with Dex was established. A decrease in the number of striated myotubes was also observed in the tissues treated with Dex. Treatment with 8 ng/mL Insulin-like Growth Factor (IGF-I) for 24 h significantly increased the contractile force of the Dex-induced atrophic tissues. The same treatment, though, had no impact on the force of the normal tissues. Thus, it is envisaged that the atrophic skeletal muscle tissues induced by Dex can be used for drug screening against atrophy.

DOI： 10.3390/bioengineering4020056

Open Access

Image-based cell quality evaluation to detect irregularities under same culture process of human induced pluripotent stem cells.

Nagasaka R, Gotou Y, Yoshida K, Kanie K, Shimizu K, Honda H, Kato R

Journal of bioscience and bioengineering 123 巻 ( 5 ) 頁： 642-650 2017年5月

Optimization of albumin secretion and metabolic activity of cytochrome P450 1A1 of human hepatoblastoma HepG2 cells in multicellular spheroids by controlling spheroid size Open Access

Nishikawa T., Tanaka Y., Nishikawa M., Ogino Y., Kusamori K., Mizuno N., Mizukami Y., Shimizu K., Konishi S., Takahashi Y., Takakura Y.

Biological and Pharmaceutical Bulletin 40 巻 ( 3 ) 頁： 334 - 338 2017年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biological and Pharmaceutical Bulletin

Multicellular spheroids are useful as three-dimensional cell culture systems and for cell-based therapies. Their successful application requires an understanding of the consequences of spheroid size for cellular functions. In the present study, we prepared multicellular spheroids of different sizes using the human hepatoblastoma HepG2 cells, as hepatocytes are frequently used for in vitro drug screening and cell-based therapy. Precise polydimethylsiloxane-based microwells with widths of 360, 450, 560, and 770 μm were fabricated using a micromolding technique. Incubation of HepG2 cells in cell culture plates containing the microwells resulted in the formation of HepG2 spheroids with average diameters of 195, 320, 493, and 548 μm. The cell number per spheroid positively correlated with its diameter, and the viability of HepG2 cells was 94% or above for all samples. The smallest HepG2 spheroids showed the highest albumin secretion. On the other hand, the metabolic activity of 7-ethoxyresorufin, a fluorometric substrate for CYP1A1, increased with increasing spheroid size. These results indicate that controlling spheroid size is important when preparing HepG2 spheroids and that the size of HepG2 spheroids greatly influences the cellular function of HepG2 cells in the spheroids.

DOI： 10.1248/bpb.b16-00833

Open Access