博士（工学） 課程 早稲田大学 知能機械学・機械システム
Haraguchi, Yuji;Shimizu, Tatsuya;Sasagawa, Tadashi;Sekine, Hidekazu;Sakaguchi, Katsuhisa;Kikuchi, Tetsutaro;Sekine, Waki;Sekiya, Sachiko;Yamato, Masayuki;Umezu, Mitsuo;Okano, Teruo
NATURE PROTOCOLS7(5)p.850 - 8582012年-2012年
Sakaguchi, Katsuhisa;Shimizu, Tatsuya;Horaguchi, Shigeto;Sekine, Hidekazu;Yamato, Masayuki;Umezu, Mitsuo;Okano, Teruo
概要：:In vitro scaling up of bioengineered tissues is known to be limited by diffusion issues, specifically a lack of vasculature. Here, we report a new strategy for preserving cell viability in three-dimensional tissues using cell sheet technology and a perfusion bioreactor having collagen-based microchannels. When triple-layer cardiac cell sheets are incubated within this bioreactor, endothelial cells in the cell sheets migrate to vascularize in the collagen gel, and finally connect with the microchannels. Medium readily flows into the cell sheets through the microchannels and the newly developed capillaries, while the cardiac construct shows simultaneous beating. When additional triple-layer cell sheets are repeatedly layered, new multi-layer construct spontaneously integrates and the resulting construct becomes a vascularized thick tissue. These results confirmed our method to fabricate in vitro vascularized tissue surrogates that overcomes engineered-tissue thickness limitations. The surrogates promise new therapies for damaged organs as well as new in vitro tissue models.
Sekine, Hidekazu;Shimizu, Tatsuya;Sakaguchi, Katsuhisa;Dobashi, Izumi;Wada, Masanori;Yamato, Masayuki;Kobayashi, Eiji;Umezu, Mitsuo;Okano, Teruo
Sakaguchi, Katsuhisa;Shimizu, Tatsuya;Okano, Teruo
JOURNAL OF CONTROLLED RELEASE205p.83 - 882015年-2015年
概要：:Construction of three-dimensional (3D) tissues with pre-isolated cells is a promising achievement for novel medicine and drug-discovery research. Our laboratory constructs 3D tissues with an innovative and unique method for layering multiple cell sheets. Cell sheets maintain a high-efficiently regenerating function, because of the higher cell density and higher transplantation efficiency, compared to other cell-delivery methods. Cell sheets have already been applied in clinical applications for regenerative medicine in treating patients with various diseases. Therefore, in our search to develop a more efficient treatment with cell sheets, we are constructing 3D tissues by layering cell sheets. Native animal tissues and organs have an abundance of capillaries to supply oxygen and nutrients, and to remove waste molecules. In our investigation of vascularized cardiac cell sheets, we have found that endothelial cells within cell sheets spontaneously form blood vessel networks as in vivo capillaries. To construct even thicker 3D tissues by layering multiple cell sheets, it is critical to have a medium or blood flow within the vascular networks of the cell sheets. Therefore, to perfuse medium or blood in the cell sheet vascular network to maintain the viability of all cells, we developed two types of vascular beds; (1) a femoral muscle-based vascular bed, and (2) a synthetic collagen gel-based vascular bed. Both vascular beds successfully provide the critical flow of culture medium, which allows 12-layer cell sheets to survive. Such bioreactor systems, when combined with cell sheet engineering techniques, have produced functional vascularized 3D tissues. Here we explain and discuss the various processes to obtain vascular networks by properly connecting cell sheets and the engineering of 3D tissues.
日本画像学会誌55(1)p.83 - 872016年-2016年
Chang, Dehua; Chang, Dehua; Shimizu, Tatsuya; Haraguchi, Yuji; Gao, Shuai; Sakaguchi, Katsuhisa; Umezu, Mitsuo; Yamato, Masayuki; Liu, Zhongmin; Okano, Teruo
概要：© 2015 Chang et al. Multilayered cell sheets have been produced from bone marrow-derived mesenchymal stem cells (MSCs) for investigating their adhesion properties onto native porcine heart tissue. Once MSCs reached confluence after a 7-day culture on a temperature-responsive culture dish, a MSCs monolayer spontaneously detached itself from the dish, when the culture temperature was reduced from 37 to 20°C. The basal extracellular matrix (ECM) proteins of the single cell sheet are preserved, because this technique requires no proteolytic enzymes for harvesting cell sheet, which become a basic building block for assembling a multilayer cell sheet. The thickness of multilayered cell sheets made from three MSC sheets was found to be approximately 60 μm. For investigating the adhesion properties of the basal and apical sides, the multilayered cell sheets were transplanted onto the surface of the heart's left ventricle. Multilayered cell sheets were histological investigated at 15, 30, 45 and 60 minutes after transplantation by hematoxylin eosin (HE) and azan dyes to determine required time for the adhesion of the multilayered sheets following cell-sheet transplantation. The results showed that only the basal side of multilayered cell sheets significantly enhanced the sheets adhesion onto the surface of heart 30 minutes after transplantation. This study concluded that (1) cell sheets had to be transplanted with its basal side onto the surface of heart tissue and (2) at least 30 minutes were necessary for obtaining the histological adhesion of the sheets to the heart tissue. This study provided clinical evidence and parameters for the successful application of MSC sheets to the myocardium and allowed cell sheet technology to be adapted clinical cell-therapy for myocardial diseases.
Takehara, Hiroaki; Takehara, Hiroaki; Sakaguchi, Katsuhisa; Kuroda, Masatoshi; Muraoka, Megumi; Itoga, Kazuyoshi; Okano, Teruo; Shimizu, Tatsuya
概要：© 2015 IOP Publishing Ltd. Cellular self-assembly based on cell-to-cell communication is a well-known tissue organizing process in living bodies. Hence, integrating cellular self-assembly processes into tissue engineering is a promising approach to fabricate well-organized functional tissues. In this research, we investigated the capability of endothelial cells (ECs) to control shape and position of vascular formation using arbitral-assembling techniques in three-dimensional engineered tissues. To quantify the degree of migration of ECs in endothelial network formation, image correlation analysis was conducted. Positive correlation between the original positions of arbitrarily assembled ECs and the positions of formed endothelial networks indicated the potential for controlling shape and position of vascular formations in engineered tissues. To demonstrate the feasibility of controlling vascular formations, engineered tissues with vascular networks in triangle and circle patterns were made. The technique reported here employs cellular self-assembly for tissue engineering and is expected to provide fundamental beneficial methods to supply various functional tissues for drug screening and regenerative medicine.
田中 龍一郎;上原 嘉宏;坂口 勝久;梅津 信二郎
IIP情報・知能・精密機器部門講演会講演論文集2016p."H - 3-2-1"-"H-3-2-2"2016年03月-2016年03月
概要：Gelatin is useful biomaterials for biofabrication. The property of gelatin is unique. The state is changed to sol or gel by temperature. Utilizing the property of gelatin, we are able to fabricate cave for artificial Vessels in biodevices. Therefore, micro gelatin fibers are useful devices for fabrication of artifical vessles. In this paper, we made a mold for fabrication of micro gelatin fibers. We used PTFE for molds, because it has non-adhesive. Then, we made micro gelatin fibers which were 20〜100 μm in width utilizing the mold. Machining marks of the mold were transcribed on the surface of micro gelatin fibers. We are able to fabricate cave with arbitrary shape for artificial vessels utilizing micro gelatin fibers.
坂口 勝久;清水 達也;松浦 勝久;大和 雅之;梅津 光生;岡野 光夫
生体医工学54(27)p.S167 - S1672016年-2016年
田中 龍一郎;新井 隆史;上原 嘉宏;坂口 勝久;梅津 信二郎
生体医工学54(27)p.S165 - S1652016年-2016年
Gelatin has unique properties. The form is changed by temperature. In addition, the melting point is around 37 degree C. For these reasons, gelatin is useful for biofabrication. We are able to fabricate biodevices of biomaterials with cave utilizing gelatin. In this study, we spun gelatin fibers utilizing three methods. We were able to fabricate micro gelatin fibers which diameter was 20~200 μm. Each methods have merits and demerits. Therefore, utilizing three methods, we were able to fabricate complex structures of micro gelatin fibers.
Haraguchi, Yuji; Kagawa, Yuki; Kagawa, Yuki; Kagawa, Yuki; Sakaguchi, Katsuhisa; Sakaguchi, Katsuhisa; Matsuura, Katsuhisa; Shimizu, Tatsuya; Okano, Teruo
概要：© 2017 The Author(s).In this paper, we report an in vitro co-culture system that combines mammalian cells and algae, Chlorococcum littorale, to create a three-dimensional (3-D) tissue. While the C2C12 mouse myoblasts and rat cardiac cells consumed oxygen actively, intense oxygen production was accounted for by the algae even in the co-culture system. Although cell metabolism within thicker cardiac cell-layered tissues showed anaerobic respiration, the introduction of innovative co-cultivation partially changed the metabolism to aerobic respiration. Moreover, the amount of glucose consumption and lactate production in the cardiac tissues and the amount of ammonia in the culture media decreased significantly when co-cultivated with algae. In the cardiac tissues devoid of algae, delamination was observed histologically, and the release of creatine kinase (CK) from the tissues showed severe cardiac cell damage. On the other hand, the layered cell tissues with algae were observed to be in a good histological condition, with less than one-fifth decline in CK release. The co-cultivation with algae improved the culture condition of the thicker tissues, resulting in the formation of 160 μm-thick cardiac tissues. Thus, the present study proposes the possibility of creating an in vitro "symbiotic recycling system" composed of mammalian cells and algae.
Arai, Takafumi; Tanaka, Ryuichiro; Sakaguchi, Katsuhisa; Umezu, Shinjiro
Artificial Life and Roboticsp.1 - 62016年12月-2016年12月
概要：© 2016 ISAROBBiotechnology has drastically been advanced by the development of iPS and ES cells, which are representative forms induced pluripotent stem cells. In the micro/nano bio field, the development of cells and Taylor-made medicine for a potential treatment of incurable diseases has been a center of attention. The melting point of gelatin is between 25 and 33 °C, and the sol–gel transition occurs in low temperature. This makes the deformation of this useful biomaterial easy. The examples of gelatin fiber applications are suture threads, blood vessel prosthesis, cell-growth-based materials, filter materials, and many others. Because the cell size differs depending on the species and applications, it is essential to fabricate gelatin fibers of different diameters. In this paper, we have developed a fabrication method for gelatin fibers the coacervation method. We fabricated narrow gelatin fibers having a diameter over 10 μm.
Hasegawa, Akiyuki; Haraguchi, Yuji; Oikaze, Hirotoshi; Kabetani, Yasuhiro; Sakaguchi, Katsuhisa; Sakaguchi, Katsuhisa; Shimizu, Tatsuya
Journal of Biomedical Materials Research - Part B Applied Biomaterials105(3)p.481 - 4882017年04月-2017年04月
概要：© 2015 Wiley Periodicals, Inc.Optical coherence tomography (OCT) is a valuable tool in the cross-sectional observation/analysis of three-dimensional (3-D) biological tissues, and that histological observation is important clinically. However, the resolution of the technology is approximately 10–20 μm. In this study, optical coherence microscopy (OCM), a tomographic system combining OCT technology with a microscopic technique, was constructed for observing cells individually with a resolution at the submicrometer level. Cells and 3-D tissues fabricated by cell sheet technology were observed by OCM. Importantly, the cell nuclei and cytoplasm could be clearly distinguished, and the time-dependent dynamics of cell-sheet tissues could be observed in detail. Additionally, the 3-D migration of cells in the bioengineered tissue was also detected using OCM and metal-labeled cells. Bovine aortic endothelial cells, but not NIH3T3 murine embryonic skin fibroblasts, actively migrated within the 3-D tissues. This study showed that the OCM system would be a valuable tool in the fields of cell biology, tissue engineering, and regenerative medicine. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 481–488, 2017.
Tanaka, Ryu ichiro; Sakaguchi, Katsuhisa; Umezu, Shinjiro; Umezu, Shinjiro
Artificial Life and Roboticsp.1 - 52017年01月-2017年01月
概要：© 2017 ISAROBGelatin is useful for biofabrication, because it can be used for cell scaffolds and it has unique properties. Therefore, we attempted to fabricate biodevices of gelatin utilizing micro 3D printer which is able to print with high precision. However, it has been difficult to fabricate 3D structure of gelatin utilizing 3D printer, because a printed gelatin droplet on the metal plate electrode would spread before solidification. To clear this problem, we developed a new experimental set-up with a peltier device that can control temperature of the impact point. At an impact point temperature of 80 °C, the spreading of printed gelatin droplets was prevented. Therefore, we were able to print a ball gelatin. In addition, we were able to print a narrower gelatin line than at an impact point temperature of 20 °C.
Sakaguchi Katsuhisa;Arai Takafumi;Shimizu Tatsuya;Umezu Shinjiro
Jpn. J. Appl. Phys.56(5)2017年04月-2017年04月
概要：Tissues engineered utilizing biofabrication techniques have recently been the focus of much attention, because these bioengineered tissues have great potential to improve the quality of life of patients with various hard-to-treat diseases. Most tissues contain micro-tubular structures including blood vessels, lymphatic vessels, and bile canaliculus. Therefore, we bioengineered a micro diameter tube using alginate gel to coat the core gelatin gel. Micro-gelatin fibers were fabricated by the coacervation method and then coated with a very thin alginate gel layer by dipping. A micro diameter alginate tube was produced by dissolving the core gelatin gel. Consequently, these procedures led to the formation of micro-alginate gel tubes of various shapes and sizes. This biofabrication technique should contribute to tissue engineering research fields.
Alshareeda, Alaa T.; Alshareeda, Alaa T.; Sakaguchi, Katsuhisa; Sakaguchi, Katsuhisa; Abumaree, Mohammed; Abumaree, Mohammed; Mohd Zin, Nur Khatijah; Mohd Zin, Nur Khatijah; Shimizu, Tatsuya
概要：© 2017 Alshareeda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background: Hepatocellular carcinoma (HCC) is considered the 3rd leading cause of death by cancer worldwide with the majority of patients were diagnosed in the late stages. Currently, there is no effective therapy. The selection of an animal model that mimics human cancer is essential for the identification of prognostic/predictive markers, candidate genes underlying cancer induction and the examination of factors that may influence the response of cancers to therapeutic agents and regimens. In this study, we developed a HCC nude rat models using cell sheet and examined the effect of human stromal cells (SCs) on the development of the HCC model and on different liver parameters such as albumin and urea. Methods: Transplanted cell sheet for HCC rat models was fabricated using thermo-responsive culture dishes. The effect of human umbilical cord mesenchymal stromal cells (UC-MSCs) and human bone marrow mesenchymal stromal cells (BM-MSCs) on the developed tumour was tested. Furthermore, development of tumour and detection of the liver parameter was studied. Additionally, angiogenesis assay was performed using Matrigel. Results: HepG2 cells requires five days to form a complete cell sheet while HepG2 co-cultured with UC-MSCs or BM-MSCs took only three days. The tumour developed within 4 weeks after transplantation of the HCC sheet on the liver of nude rats. Both UC-MSCs and BM-MSCs improved the secretion of liver parameters by increasing the secretion of albumin and urea. Comparatively, the UC-MSCs were more effective than BM-MSCs, but unlike BM-MSCs, UC-MSCs prevented liver tumour formation and the tube formation of HCC. Conclusions: Since this is a novel study to induce liver tumour in rats using hepatocellular carcinoma sheet and stromal cells, the data obtained suggest that cell sheet is a fast and easy technique to develop HCC models as well as UC-MSCs have therapeutic potential for liver diseases. Additionally, the data procured indicates that stromal cells enhanced the fabrication of HepG2 cell sheets. This provides the foundation for future research using stromal cells in preclinical and clinical investigations.
Sakaguchi, Katsuhisa; Arai, Takafumi; Shimizu, Tatsuya; Umezu, Shinjiro
Japanese Journal of Applied Physics56(5)2017年05月-2017年05月
概要：© 2017 The Japan Society of Applied Physics. Tissues engineered utilizing biofabrication techniques have recently been the focus of much attention, because these bioengineered tissues have great potential to improve the quality of life of patients with various hard-to-treat diseases. Most tissues contain micro-tubular structures including blood vessels, lymphatic vessels, and bile canaliculus. Therefore, we bioengineered a micro diameter tube using alginate gel to coat the core gelatin gel. Micro-gelatin fibers were fabricated by the coacervation method and then coated with a very thin alginate gel layer by dipping. A micro diameter alginate tube was produced by dissolving the core gelatin gel. Consequently, these procedures led to the formation of micro-alginate gel tubes of various shapes and sizes. This biofabrication technique should contribute to tissue engineering research fields.
梅津 信二郎, 坂口 勝久, 秋元 渓
|生命医科学実験I [S Grade]||先進理工学部||2020||春学期|
|Research on Regenerative Medical Engineering and its Application||大学院先進理工学研究科||2020||通年|
|Practical Medical Engineering||大学院先進理工学研究科||2020||春学期|
|Seminar on Regenerative Medical Engineering A||大学院先進理工学研究科||2020||春学期|
|Seminar on Regenerative Medical Engineering B||大学院先進理工学研究科||2020||秋学期|
|Seminar on Regenerative Medical Engineering C||大学院先進理工学研究科||2020||春学期|
|Seminar on Regenerative Medical Engineering D||大学院先進理工学研究科||2020||秋学期|
|Master's Thesis (Department of Integrative Bioscience and Biomedical Engineering)||大学院先進理工学研究科||2020||通年|