The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabrication system for functional 3D tissues by stacking cell sheets of confluent cultured cells detached from a temperature-responsive culture dish. Here we describe the protocols for the fabrication of 3D tissues by cell sheet engineering. Three-dimensional cardiac tissues fabricated by stacking cardiac cell sheets pulsate spontaneously, synchronously and macroscopically. Via this protocol, it is also possible to fabricate other tissues, such as 3D tissue including capillary-like prevascular networks, from endothelial cells sandwiched between layered cell sheets. Cell sheet stacking technology promises to provide in vitro tissue/organ models and more effective therapies for curing tissue/organ failures.

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 temperatureresponsive 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 myocardial tissues, these patch-like tissue grafts were transplanted onto infarcted rat hearts. Four weeks after transplantation, recovery of cardiac function could be significantly improved by increasing the EC densities within the engineered myocardial tissues. Additionally, when the EC-positive cardiac tissues were transplanted to myocardial infarction models, we observed significantly greater numbers of capillaries in the grafts as compared with the EC-negative cell sheets. Finally, blood vessels originating from the engineered EC-positive cardiac tissues bridged into the infarcted myocardium to connect with capillaries of the host heart. Conclusions-In vitro engineering of 3-dimensional cardiac tissues with preformed EC networks that can be easily connected to host vessels can contribute to the reconstruction of myocardial tissue grafts with a high potential for cardiac function repair. These results indicate that neovascularization can contribute to improved cardiac function after the transplantation of engineered cardiac tissues. (Circulation. 2008;118[suppl 1]:S145-S152.)

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Aims/Introduction: Adipose-derived mesenchymal stem cell (ASC) transplantation is a promising therapy for diabetic nephropathy (DN). However, intravascular administration of ASCs is associated with low engraftment in target organs. Therefore, we considered applying the cell sheet technology to ASCs. In this study, ASC sheets were directly transplanted into the kidneys of a DN rat model, and therapeutic consequences were analyzed. Materials and Methods: Adipose-derived mesenchymal stem cells were isolated from adipose tissues of 7-week-old enhanced green fluorescent protein rats, and ASC sheets were prepared using a temperature-responsive culture dish. A DN rat model was established from 5-week-old Spontaneously Diabetic Torii fatty rats. Seven-week-old DN rats (n = 21) were assigned to one of the following groups: sham-operated (n = 6); ASC suspension (6.0 9 10 6 cells/mL) administered intravenously (n = 7); six ASC sheets transplanted directly into the kidney (n = 8). The therapeutic effect of the cell sheets was determined based on urinary biomarker expression and histological analyses. Results: The ASC sheets survived under the kidney capsule of the DN rat model for 14 days after transplantation. Furthermore, albuminuria and urinary tumor necrosis factor-a levels were significantly lower in the ASC sheets transplanted directly into the kidney group than in the sham-operated and ASC suspension administered intravenously groups (P < 0.05). Histologically, the ASC sheets transplanted directly into the kidney group presented mild atrophy of the proximal tubule and maintained the renal tubular structure. Conclusions: Transplantation of ASC sheets directly into the kidney improved transplantation efficiency and suppressed renal injury progression. Therefore, the ASC sheet technology might be a promising novel treatment for DN.Although strict glycemic control is reported to suppress the progression of DN 6,7 , there is no treatment to stop DN progression. Recently, mesenchymal stem cell (MSC) transplantation has received substantial attention as a therapy for DN. MSCs can differentiate into adipogenic and osteogenic cells 8 , and possess clinically useful properties, such as low antigenicity 9,10 and paracrine effects through cytokines 11,12 . In particular, adipose-derived mesenchymal stem cells (ASCs) represent an attractive cell source owing to their abundance in the body 13,14 .In animal experiments on DN, intravascular administration of ASC suspensions was shown to suppress the progression of

As proposed in the late 1980s by Langer and Vacanti, the ultimate goal of tissue engineering is the development of structures that can be used to treat or replace damaged or diseased organs and tissues. For the regeneration of various organs such as the heart, liver, and kidney, the development of adequate vascular networks within the engineered tissues remains a significant obstacle in the formation of cell‐dense structures that resemble the native parenchyma. While tissue engineering using biodegradable scaffolds has been successful in the re‐creation of tissues where extracellular matrix is abundant, we have developed cell‐sheet‐based tissue engineering for the construction of tissues using laminar assemblies of cells harvested from temperature‐responsive culture dishes. Using cell sheet engineering, we present new strategies for the development of organ‐like tissue structures containing well‐organized vascular networks.