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.

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A major challenge of tissue engineering is the creation of three-dimensional (3D) and functional tissues. However, the tissue ischemic environments make the production of thicker 3D tissues difficult. For evaluating the thickness limitation of tissue, the cell viability and metabolism of a novel in vitro 3D tissue model, which is composed of multilayered cell sheets, were investigated. Human endometrial-derived mesenchymal cells (EMC) were cultured on temperature-responsive culture dishes. Confluently cultured EMCs were harvested as an intact contiguous cell sheet only by lowering temperature. The obtained cell sheets were successfully layered into 3D cell-dense tissues and cultured in vitro for 7 days. Glucose consumption and lactate production in the culture media increased in accordance with the number of layers (single to triple). Histological analyses and cell viability assays showed that viable tissues were found in single-to triple-layered cell sheets and damaged tissues in over quadruple layers. The results concluded that the thickness limitation of layered cell sheets was approximately 40 mm, which was the thickness of triple-layered cell sheets. Importantly, when multi-layered cell sheets were cultured on porous membranes, the adhesion among cell sheets and cell viability were improved, resulting in successful fabricating thicker tissues (~100 mm) than that on normal culture dishes. The metabolic analyses showed that multi-layered cell sheets rely on their anaerobic metabolism, indicating that supplying nutrients rather than oxygen through both upper and bottom tissue surfaces improved the cell viability in 3D tissues. ` `Citation: Sekine W, Haraguchi Y, Shimizu T, Umezawa A, Okano T (2011) Thickness limitation and cell viability of multi-layered cell sheets and overcoming the diffusion limit by a porous-membrane culture insert. J Biochip Tissue chip S1:007.

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