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Teruo Okano - One of the best experts on this subject based on the ideXlab platform.
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tissue engineered myoblast sheets improved cardiac function sufficiently to discontinue lvas in a patient with dcm report of a case
Surgery Today, 2012Co-Authors: Yoshiki Sawa, Tatsuya Shimizu, Shigeru Miyagawa, Taichi Sakaguchi, Tomoyuki Fujita, Akifumi Matsuyama, Atsuhiro Saito, Teruo OkanoAbstract:Dilated cardiomyopathy (DCM) is a heart muscle disease characterized by progressive heart failure, and is a leading cause of mortality and morbidity. Recently, cellular therapy for end-stage heart failure has been emerging. We herein report a 56-year-old male who received a transplant of autologous myoblast sheets manufactured in temperature-responsive Culture Dishes. His clinical condition improved markedly, leaving him without any arrhythmia and able to discontinue using a left ventricular assist system and avoid cardiac transplantation. These findings suggest that cellular therapy using myoblast sheets is a promising new strategy for treating patients with end-stage DCM. This method might be an effective alternative to heart transplantation in the near future.
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endothelial cell coCulture within tissue engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts
Circulation, 2008Co-Authors: Hidekazu Sekine, Sachiko Sekiya, Joseph Yang, Hiromi Kurosawa, Kyoko Hobo, Tatsuya Shimizu, Masayuki Yamato, Eiji Kobayashi, Teruo OkanoAbstract:Background— Regenerative therapies, including myocardial tissue engineering, have been pursued as a new possibility to repair the damaged myocardium, and previously the transplantation of layered cardiomyocyte sheets has been shown to be able to improve cardiac function after myocardial infarction. We examined the effects of promoting neovascularization by controlling the densities of coCultured endothelial cells (ECs) within engineered myocardial tissues created using our cell sheet-based tissue engineering approach. Methods and Results— Neonatal rat cardiomyocytes were coCultured with GFP-positive rat-derived ECs on temperature-responsive Culture Dishes. CoCultured ECs formed cell networks within the cardiomyocyte sheets, which were preserved during cell harvest from the Dishes using simple temperature reduction. We also observed significantly increased in vitro production of vessel-forming cytokines by the EC-positive cardiac cell sheets. After layering of 3 cardiac cell sheets to create 3-dimensional ...
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electrical coupling of cardiomyocyte sheets occurs rapidly via functional gap junction formation
Biomaterials, 2006Co-Authors: Yuji Haraguchi, Tatsuya Shimizu, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:Abstract Previously, we have successfully created pulsatile myocardial tissue grafts using our novel technology, “cell sheet engineering”, that layers cell sheets fabricated on temperature-responsive Culture Dishes to form three-dimensional (3-D) structures. Electrical coupling is established between layered neonatal rat cardiomyocyte sheets, resulting in the synchronized beating of 3-D myocardial tissues. However, the mechanism by which these layered cardiomyocyte sheets communicate electrically is not well-understood. In this study, we used a multiple-electrode extracellular recording system and demonstrated that bilayer cardiomyocyte sheets coupled electrically with slight delays 34±2 min (mean±SEM) after layering. These delays gradually decreased and the electrical actions of layered cell sheets were completely coupled 46±3 min (mean±SEM) after initial layering. Immunohistological analysis showed that connexin43, a gap junction (GJ)-related protein, existed not only at cell-to-cell interfaces but also on the free cell membrane in the cardiomyocyte sheet. Additionally, neither connexin40 nor connexin45, but only connexin43 was detected between bilayer cardiomyocyte sheets within 30 min after layering. Dye transfer assay demonstrated that the exchange of small molecules via GJs occurred within 30 min. The cell sheet manipulation technique using the temperature-responsive Culture Dishes has substantial advances and the exciting potential in the fields of cell and tissue physiology, as well as tissue engineering.
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bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential
Biochemical and Biophysical Research Communications, 2006Co-Authors: Sachiko Sekiya, Tatsuya Shimizu, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:Abstract Previously, we have demonstrated the long-term survival of myocardial cell sheet constructs in vivo, with microvascular network formation throughout the engineered tissues. The understanding and control of these vascularization processes are a key factor for creating thicker functional tissues. Here, we show that cardiac cell sheets express angiogenesis-related genes and form endothelial cell networks in Culture. After non-invasive harvest and stacking of cell sheets using temperature-responsive Culture Dishes, these endothelial cell networks are maintained and result in neovascularization upon in vivo transplantation. Interestingly, we also discovered that all of the graft vessels are derived from the grafts themselves and these vessels migrate to connect with the host vasculature. Finally, blood vessel formation within the grafts can be controlled by changing the ratio of endothelial cells. In conclusion, myocardial tissue grafts engineered with cell sheet technology have their own inherent potential for the in vivo neovascularization that can be regulated in vitro.
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cardiomyocyte bridging between hearts and bioengineered myocardial tissues with mesenchymal transition of mesothelial cells
Journal of Heart and Lung Transplantation, 2006Co-Authors: Hidekazu Sekine, Tatsuya Shimizu, Eiji Kobayashi, Seiichi Kosaka, Teruo OkanoAbstract:Background For the reconstruction of 3-dimensional (3D) tissues, we exploited an original method of tissue engineering that layers individual cell sheets harvested from temperature-responsive Culture Dishes. Stacked cardiomyocyte sheets demonstrated electrical and morphologic communication, resulting in synchronously beating myocardial tissue. When these bioengineered 3D tissue grafts are transplanted onto damaged hearts, gap junction communication between graft and host is likely critical for synchronized beating and functional improvement. In this study, these graft-to-heart morphologic communications were examined. Methods Neonatal rat cardiomyocyte sheets were harvested from temperature-responsive Culture Dishes and layered to create 3D tissues. These constructs were then transplanted onto infarcted rat hearts. Histologic analyses and transmission electron microscopy (TEM) were performed to examine morphologic communications. The passage of small molecules through functional gap junctions was also detected using a dye-transfer assay. Results Transplanted cardiomyocytes bridged between the grafts and hearts in intact areas. Connexin-43 staining and TEM revealed the existence of gap junctions and intercalated disks between the bridging cardiomyocytes. Furthermore, it was confirmed that a low-molecule fluorescent dye, calcein, was transferred from the grafts to the hearts via the bridging cardiomyocytes. Immunohistochemistry with anti-intercellular adhesion molecule-1 antibodies revealed that mesothelial cells in the epicardium scattered and transdifferentiated into mesenchymal cells between the graft and host. Conclusions The direct attachment of layered cardiomyocyte sheets on the heart surface promotes mesothelial cell transdifferentiation and cardiomyocyte bridging, leading to functional communication via gap junctions. These results indicate that these bioengineered myocardial tissues may improve damaged heart function via synchronized beating.
Masayuki Yamato - One of the best experts on this subject based on the ideXlab platform.
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Surface design of antibody-immobilized thermoresponsive cell Culture Dishes for recovering intact cells by low-temperature treatment
Journal of Biomedical Materials Research Part A, 2013Co-Authors: Jun Kobayashi, Masayuki Yamato, Masaki Hayashi, Takahiro Ohno, Masanori Nishi, Yoshinori Arisaka, Yoshinori Matsubara, Hiroshi Kakidachi, Yoshikatsu Akiyama, Akihiro HoriiAbstract:Antibody-immobilized thermoresponsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [poly(IPAAm-co-CIPAAm)]-grafted cell Culture surfaces were designed to enhance both the initial adhesion of weakly adhering cells and the ability of cells to detach in response to low temperature through the regulation of affinity binding between immobilized antibodies and antigens on the cellular surface. Ty-82 cells and neonatal normal human dermal fibroblasts (NHDFs), which express CD90 on the cell surface, adhered to anti-CD90 antibody-immobilized thermoresponsive surfaces at 37°C, a condition at which the grafted thermoresponsive polymer chains shrank. Adherent Ty-82 cells were detached from the surfaces by lowering the temperature to 20°C and applying external forces, such as pipetting, whereas Cultured NHDF sheets spontaneously detached themselves from the surface in response to reduced temperature alone. When the temperature was decreased to 20°C, the swelling of grafted thermoresponsive polymer chains weakened the affinity binding between immobilized antibody and antigen on the cells due to the increasing steric hindrance of the polymer chains around the antigen-recognition site of the immobilized antibodies. No contamination was detected on cells harvested from covalently immobilized antibodies on the Culture surfaces by low-temperature treatment, whereas a carryover of the antibody and avidin from the avidin-biotin binding surface was observed. Furthermore, the initial adhesion of adipose tissue-derived cells, which adhere weakly to PIPAAm-grafted surfaces, was enhanced on the antibody-immobilized thermoresponsive surfaces. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3883–3893, 2014.
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endothelial cell coCulture within tissue engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts
Circulation, 2008Co-Authors: Hidekazu Sekine, Sachiko Sekiya, Joseph Yang, Hiromi Kurosawa, Kyoko Hobo, Tatsuya Shimizu, Masayuki Yamato, Eiji Kobayashi, Teruo OkanoAbstract:Background— Regenerative therapies, including myocardial tissue engineering, have been pursued as a new possibility to repair the damaged myocardium, and previously the transplantation of layered cardiomyocyte sheets has been shown to be able to improve cardiac function after myocardial infarction. We examined the effects of promoting neovascularization by controlling the densities of coCultured endothelial cells (ECs) within engineered myocardial tissues created using our cell sheet-based tissue engineering approach. Methods and Results— Neonatal rat cardiomyocytes were coCultured with GFP-positive rat-derived ECs on temperature-responsive Culture Dishes. CoCultured ECs formed cell networks within the cardiomyocyte sheets, which were preserved during cell harvest from the Dishes using simple temperature reduction. We also observed significantly increased in vitro production of vessel-forming cytokines by the EC-positive cardiac cell sheets. After layering of 3 cardiac cell sheets to create 3-dimensional ...
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electrical coupling of cardiomyocyte sheets occurs rapidly via functional gap junction formation
Biomaterials, 2006Co-Authors: Yuji Haraguchi, Tatsuya Shimizu, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:Abstract Previously, we have successfully created pulsatile myocardial tissue grafts using our novel technology, “cell sheet engineering”, that layers cell sheets fabricated on temperature-responsive Culture Dishes to form three-dimensional (3-D) structures. Electrical coupling is established between layered neonatal rat cardiomyocyte sheets, resulting in the synchronized beating of 3-D myocardial tissues. However, the mechanism by which these layered cardiomyocyte sheets communicate electrically is not well-understood. In this study, we used a multiple-electrode extracellular recording system and demonstrated that bilayer cardiomyocyte sheets coupled electrically with slight delays 34±2 min (mean±SEM) after layering. These delays gradually decreased and the electrical actions of layered cell sheets were completely coupled 46±3 min (mean±SEM) after initial layering. Immunohistological analysis showed that connexin43, a gap junction (GJ)-related protein, existed not only at cell-to-cell interfaces but also on the free cell membrane in the cardiomyocyte sheet. Additionally, neither connexin40 nor connexin45, but only connexin43 was detected between bilayer cardiomyocyte sheets within 30 min after layering. Dye transfer assay demonstrated that the exchange of small molecules via GJs occurred within 30 min. The cell sheet manipulation technique using the temperature-responsive Culture Dishes has substantial advances and the exciting potential in the fields of cell and tissue physiology, as well as tissue engineering.
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bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential
Biochemical and Biophysical Research Communications, 2006Co-Authors: Sachiko Sekiya, Tatsuya Shimizu, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:Abstract Previously, we have demonstrated the long-term survival of myocardial cell sheet constructs in vivo, with microvascular network formation throughout the engineered tissues. The understanding and control of these vascularization processes are a key factor for creating thicker functional tissues. Here, we show that cardiac cell sheets express angiogenesis-related genes and form endothelial cell networks in Culture. After non-invasive harvest and stacking of cell sheets using temperature-responsive Culture Dishes, these endothelial cell networks are maintained and result in neovascularization upon in vivo transplantation. Interestingly, we also discovered that all of the graft vessels are derived from the grafts themselves and these vessels migrate to connect with the host vasculature. Finally, blood vessel formation within the grafts can be controlled by changing the ratio of endothelial cells. In conclusion, myocardial tissue grafts engineered with cell sheet technology have their own inherent potential for the in vivo neovascularization that can be regulated in vitro.
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cell sheet engineering recreating tissues without biodegradable scaffolds
Biomaterials, 2005Co-Authors: Joseph Yang, Hidekazu Sekine, Masayuki Yamato, Chinatsu Kohno, Ayako Nishimoto, Fumio Fukai, Teruo OkanoAbstract:While tissue engineering has long been thought to possess enormous potential, conventional applications using biodegradable scaffolds have limited the field's progress, demonstrating a need for new methods. We have previously developed cell sheet engineering using temperature-responsive Culture Dishes in order to avoid traditional tissue engineering approaches, and their related shortcomings. Using temperature-responsive Dishes, Cultured cells can be harvested as intact sheets by simple temperature changes, thereby avoiding the use of proteolytic enzymes. Cell sheet engineering therefore allows for tissue regeneration by either direct transplantation of cell sheets to host tissues or the creation of three-dimensional structures via the layering of individual cell sheets. By avoiding the use of any additional materials such as carrier substrates or scaffolds, the complications associated with traditional tissue engineering approaches such as host inflammatory responses to implanted polymer materials, can be avoided. Cell sheet engineering thus presents several significant advantages and can overcome many of the problems that have previously restricted tissue engineering with biodegradable scaffolds.
Akihiko Kikuchi - One of the best experts on this subject based on the ideXlab platform.
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electrical coupling of cardiomyocyte sheets occurs rapidly via functional gap junction formation
Biomaterials, 2006Co-Authors: Yuji Haraguchi, Tatsuya Shimizu, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:Abstract Previously, we have successfully created pulsatile myocardial tissue grafts using our novel technology, “cell sheet engineering”, that layers cell sheets fabricated on temperature-responsive Culture Dishes to form three-dimensional (3-D) structures. Electrical coupling is established between layered neonatal rat cardiomyocyte sheets, resulting in the synchronized beating of 3-D myocardial tissues. However, the mechanism by which these layered cardiomyocyte sheets communicate electrically is not well-understood. In this study, we used a multiple-electrode extracellular recording system and demonstrated that bilayer cardiomyocyte sheets coupled electrically with slight delays 34±2 min (mean±SEM) after layering. These delays gradually decreased and the electrical actions of layered cell sheets were completely coupled 46±3 min (mean±SEM) after initial layering. Immunohistological analysis showed that connexin43, a gap junction (GJ)-related protein, existed not only at cell-to-cell interfaces but also on the free cell membrane in the cardiomyocyte sheet. Additionally, neither connexin40 nor connexin45, but only connexin43 was detected between bilayer cardiomyocyte sheets within 30 min after layering. Dye transfer assay demonstrated that the exchange of small molecules via GJs occurred within 30 min. The cell sheet manipulation technique using the temperature-responsive Culture Dishes has substantial advances and the exciting potential in the fields of cell and tissue physiology, as well as tissue engineering.
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bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential
Biochemical and Biophysical Research Communications, 2006Co-Authors: Sachiko Sekiya, Tatsuya Shimizu, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:Abstract Previously, we have demonstrated the long-term survival of myocardial cell sheet constructs in vivo, with microvascular network formation throughout the engineered tissues. The understanding and control of these vascularization processes are a key factor for creating thicker functional tissues. Here, we show that cardiac cell sheets express angiogenesis-related genes and form endothelial cell networks in Culture. After non-invasive harvest and stacking of cell sheets using temperature-responsive Culture Dishes, these endothelial cell networks are maintained and result in neovascularization upon in vivo transplantation. Interestingly, we also discovered that all of the graft vessels are derived from the grafts themselves and these vessels migrate to connect with the host vasculature. Finally, blood vessel formation within the grafts can be controlled by changing the ratio of endothelial cells. In conclusion, myocardial tissue grafts engineered with cell sheet technology have their own inherent potential for the in vivo neovascularization that can be regulated in vitro.
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Influence of insulin immobilization to thermoresponsive Culture surfaces on cell proliferation and thermally induced cell detachment
Biomaterials, 2005Co-Authors: Hideyuki Hatakeyama, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:Temperature-responsive Culture Dishes immobilized with insulin have been fabricated and studied to shorten cell Culture periods by facilitating more rapid cell proliferation. Cells are recovered as contiguous cell sheets simply by temperature changes. Functionalized Culture Dishes were prepared by previously reported electron beam grafting copolymerization of N-isopropylacrylamide (IPAAm) with its carboxylate-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), having similar molecular structure to IPAAm but with carboxylate side chains to tissue Culture polystyrene Dishes. Insulin was then immobilized onto Culture Dishes through standard amide bond formation with CIPAAm carboxylate groups. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on these insulin-immobilized Dishes. Insulin immobilization was shown to promote cell proliferation in serum-supplemented medium. Increasing the grafted CIPAAm content on the tissue Culture surfaces reduces cell adhesion and proliferation, even though these surfaces contained increased amounts of immobilized insulin. This result implies that a discrete balance exists between the amount of CIPAAm-free carboxylate groups and immobilized insulin for optimum cell proliferative stimulation. Cells grown on the insulin-immobilized surfaces can be recovered as contiguous cell monolayers simply by lowering Culture temperature, without need for exogenous enzyme or calcium chelator additions. In conclusion, insulin-modified thermoresponsive Culture Dishes may prove useful for advanced cell Culture and tissue engineering applications since they facilitate cell proliferation, and Cultured cells can be recovered as viable contiguous monolayers by merely reducing Culture temperature.
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Control of cell adhesion and detachment using temperature and thermoresponsive copolymer grafted Culture surfaces.
Journal of biomedical materials research. Part A, 2004Co-Authors: Yukiko Tsuda, Akihiko Kikuchi, Masayuki Yamato, Yasuhisa Sakurai, Mitsuo Umezu, Teruo OkanoAbstract:The hydrophobic monomer, n-butyl methacrylate (BMA) has been incorporated into thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) to lower PIPAAm phase transition temperatures necessary for systematically regulating cell adhesion on and detachment from Culture Dishes at controlled temperatures. Poly(IPAAm-co-BMA)-grafted Dishes were prepared by electron beam irradiation methods, systematically changing BMA content in the feed. Copolymer-grafted surfaces decreased grafted polymer transition temperatures with increasing BMA content as shown by water wettabilities compared to homopolymer PIPAAm-grafted surfaces. Bovine endothelial cells readily adhered and proliferated on copolymer-grafted surfaces above collapse temperature at 37°C, finally reaching confluence. Cell sheet detachment behavior from copolymer-grafted surfaces depended on the Culture temperature and BMA content. In conclusion, cell attachment/detachment can be controlled to an arbitrary temperature by varying the content of hydrophobic monomer incorporated into PIPAAm grafted to Culture surfaces. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 70–78, 2004
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novel approach for achieving double layered cell sheets co Culture overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature responsive Culture Dishes
Journal of Biomedical Materials Research, 2002Co-Authors: Masami Harimoto, Akihiko Kikuchi, Masayuki Yamato, Motohiro Hirose, Chie Takahashi, Yuki Isoi, Teruo OkanoAbstract:Confluent human aortic endothelial cells (HAECs) Cultured on thermo-responsive Culture dish grafted with poly (N-isopropylacrylamide) were recovered as a contiguous cell sheet. The double-layered co-Culture was achieved by placing the recovered HAEC sheet onto the rat hepatocyte layer directly. The double-layered structure of HAEC and hepatocytes remained in tight contact during Culture. Hepatocytes in the layered co-Culture system with the HAEC sheet maintained the differentiated cell shape and the albumin expression for over 41 days of Culture, whereas the functions disappeared within 10 days of Culture in control hepatocytes without the HAEC sheet. The layered co-Culture of hepatocytes and the HAEC sheets, which allows for the expression of differentiated functions of hepatocyte continuously, such as liver lobule, offers a major advancement in liver tissue engineering.
Ai Kushida - One of the best experts on this subject based on the ideXlab platform.
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thermo responsive Culture Dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature
Tissue Engineering, 2001Co-Authors: Masayuki Yamato, Akihiko Kikuchi, Mika Utsumi, Ai Kushida, Chie Konno, Teruo OkanoAbstract:To develop new technology for harvesting transplantable Cultured epithelium without dispase treatment, human keratinocytes were plated on Culture Dishes grafted with a thermo-responsive polymer, poly(N-isopropylacrylamide). The grafted dish surfaces are slightly hydrophobic above 32°C, but reversibly change to hydrophilic below this temperature. According to the method of Rheinwald and Green, keratinocytes proliferated and made a multilayer on the grafted surfaces at 37°C, as on the nongrafted Culture Dishes. The multilayered keratinocyte sheets were detached from the grafted surfaces only by reducing temperature to 20°C without need for dispase. No cell remnants were observed on the Dishes. Such cell sheet detachment was not observed on nongrafted Dishes. Immunoblotting of harvested keratinocyte sheets revealed that dispase treatment disrupted E-cadherin and laminin 5, while these molecules remained intact in the keratinocyte sheets harvested by only reducing temperature from the grafted Dishes. Transmis...
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thermo responsive Culture Dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature
Tissue Engineering, 2001Co-Authors: Masayuki Yamato, Akihiko Kikuchi, Mika Utsumi, Ai Kushida, Chie Konno, Teruo OkanoAbstract:To develop new technology for harvesting transplantable Cultured epithelium without dispase treatment, human keratinocytes were plated on Culture Dishes grafted with a thermo-responsive polymer, po...
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two dimensional manipulation of differentiated madin darby canine kidney mdck cell sheets the noninvasive harvest from temperature responsive Culture Dishes and transfer to other surfaces
Journal of Biomedical Materials Research, 2001Co-Authors: Ai Kushida, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:A renal epithelial cell line, Madin–Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive Culture Dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37°C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted Dishes merely by reducing the temperature. However, during the early stage of Culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other Culture Dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-β-catenin antibody revealed that the functional cell–cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets. © 2000 John Wiley & Sons, Inc.
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two dimensional manipulation of differentiated madin darby canine kidney mdck cell sheets the noninvasive harvest from temperature responsive Culture Dishes and transfer to other surfaces
Journal of Biomedical Materials Research, 2001Co-Authors: Ai Kushida, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:A renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive Culture Dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37 degrees C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted Dishes merely by reducing the temperature. However, during the early stage of Culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other Culture Dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-beta-catenin antibody revealed that the functional cell-cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets.
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temperature responsive Culture Dishes allow nonenzymatic harvest of differentiated madin darby canine kidney mdck cell sheets
Journal of Biomedical Materials Research, 2000Co-Authors: Ai Kushida, Akihiko Kikuchi, Masayuki Yamato, Chie Konno, Yasuhisa Sakurai, Teruo OkanoAbstract:We have developed a temperature-responsive Culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted Dishes in the presence of serum at 37°C. By reducing only temperature, these cells can be harvested noninvasively from the Dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was Cultured on the Dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term Culture, similar to on ungrafted Dishes. After 1-week Culture at 37°C, trypsin digestion disrupted cell–cell junctions but failed to liberate cells from both ungrafted and grafted Dishes. However, short-term incubation at 20°C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted Dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-β-catenin antibody revealed that functional cell–cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20°C required for cell sheet detachment gradually shortened during long-term Culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted Dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 51, 216–223, 2000.
Tatsuya Shimizu - One of the best experts on this subject based on the ideXlab platform.
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creation of human cardiac cell sheets using pluripotent stem cells
Biochemical and Biophysical Research Communications, 2012Co-Authors: Katsuhisa Matsuura, Masanori Wada, Yuji Haraguchi, Fumiko Sato, Kasumi Sugiyama, Yuji Shiba, Hinako Ichikawa, Kanako Konishi, Tatsuya Shimizu, Aki TachibanaAbstract:Abstract Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets, the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study, we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor Culture for 14 days produced around 8 × 10 7 cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation, cardiomyocytes were Cultured on temperature-responsive Culture Dishes and showed spontaneous and synchronous beating, even after cell sheets were detached from Culture Dishes. Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue.
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tissue engineered myoblast sheets improved cardiac function sufficiently to discontinue lvas in a patient with dcm report of a case
Surgery Today, 2012Co-Authors: Yoshiki Sawa, Tatsuya Shimizu, Shigeru Miyagawa, Taichi Sakaguchi, Tomoyuki Fujita, Akifumi Matsuyama, Atsuhiro Saito, Teruo OkanoAbstract:Dilated cardiomyopathy (DCM) is a heart muscle disease characterized by progressive heart failure, and is a leading cause of mortality and morbidity. Recently, cellular therapy for end-stage heart failure has been emerging. We herein report a 56-year-old male who received a transplant of autologous myoblast sheets manufactured in temperature-responsive Culture Dishes. His clinical condition improved markedly, leaving him without any arrhythmia and able to discontinue using a left ventricular assist system and avoid cardiac transplantation. These findings suggest that cellular therapy using myoblast sheets is a promising new strategy for treating patients with end-stage DCM. This method might be an effective alternative to heart transplantation in the near future.
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endothelial cell coCulture within tissue engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts
Circulation, 2008Co-Authors: Hidekazu Sekine, Sachiko Sekiya, Joseph Yang, Hiromi Kurosawa, Kyoko Hobo, Tatsuya Shimizu, Masayuki Yamato, Eiji Kobayashi, Teruo OkanoAbstract:Background— Regenerative therapies, including myocardial tissue engineering, have been pursued as a new possibility to repair the damaged myocardium, and previously the transplantation of layered cardiomyocyte sheets has been shown to be able to improve cardiac function after myocardial infarction. We examined the effects of promoting neovascularization by controlling the densities of coCultured endothelial cells (ECs) within engineered myocardial tissues created using our cell sheet-based tissue engineering approach. Methods and Results— Neonatal rat cardiomyocytes were coCultured with GFP-positive rat-derived ECs on temperature-responsive Culture Dishes. CoCultured ECs formed cell networks within the cardiomyocyte sheets, which were preserved during cell harvest from the Dishes using simple temperature reduction. We also observed significantly increased in vitro production of vessel-forming cytokines by the EC-positive cardiac cell sheets. After layering of 3 cardiac cell sheets to create 3-dimensional ...
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electrical coupling of cardiomyocyte sheets occurs rapidly via functional gap junction formation
Biomaterials, 2006Co-Authors: Yuji Haraguchi, Tatsuya Shimizu, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:Abstract Previously, we have successfully created pulsatile myocardial tissue grafts using our novel technology, “cell sheet engineering”, that layers cell sheets fabricated on temperature-responsive Culture Dishes to form three-dimensional (3-D) structures. Electrical coupling is established between layered neonatal rat cardiomyocyte sheets, resulting in the synchronized beating of 3-D myocardial tissues. However, the mechanism by which these layered cardiomyocyte sheets communicate electrically is not well-understood. In this study, we used a multiple-electrode extracellular recording system and demonstrated that bilayer cardiomyocyte sheets coupled electrically with slight delays 34±2 min (mean±SEM) after layering. These delays gradually decreased and the electrical actions of layered cell sheets were completely coupled 46±3 min (mean±SEM) after initial layering. Immunohistological analysis showed that connexin43, a gap junction (GJ)-related protein, existed not only at cell-to-cell interfaces but also on the free cell membrane in the cardiomyocyte sheet. Additionally, neither connexin40 nor connexin45, but only connexin43 was detected between bilayer cardiomyocyte sheets within 30 min after layering. Dye transfer assay demonstrated that the exchange of small molecules via GJs occurred within 30 min. The cell sheet manipulation technique using the temperature-responsive Culture Dishes has substantial advances and the exciting potential in the fields of cell and tissue physiology, as well as tissue engineering.
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bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential
Biochemical and Biophysical Research Communications, 2006Co-Authors: Sachiko Sekiya, Tatsuya Shimizu, Akihiko Kikuchi, Masayuki Yamato, Teruo OkanoAbstract:Abstract Previously, we have demonstrated the long-term survival of myocardial cell sheet constructs in vivo, with microvascular network formation throughout the engineered tissues. The understanding and control of these vascularization processes are a key factor for creating thicker functional tissues. Here, we show that cardiac cell sheets express angiogenesis-related genes and form endothelial cell networks in Culture. After non-invasive harvest and stacking of cell sheets using temperature-responsive Culture Dishes, these endothelial cell networks are maintained and result in neovascularization upon in vivo transplantation. Interestingly, we also discovered that all of the graft vessels are derived from the grafts themselves and these vessels migrate to connect with the host vasculature. Finally, blood vessel formation within the grafts can be controlled by changing the ratio of endothelial cells. In conclusion, myocardial tissue grafts engineered with cell sheet technology have their own inherent potential for the in vivo neovascularization that can be regulated in vitro.
