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.

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.

A novel assay system with cell-dense three-dimensional (3D) tissue was developed for measuring the permeability of substances. In this paper, the permeabilities of various molecules containing nutrients, a cytokine, and a chemokine were examined and analyzed. A single-layered cell sheet was approximately 20 mum thick, and as the number of layers of these cell sheets increased, so did the total thickness of the tissue. The diffusion rates of glucose and pyruvic acid were reduced to approximately 30-40% by a single-layered cell sheet compared with the control without the cell sheet, and the diffusion of both substances were completely inhibited by a quadruple-layered cell sheet. The diffusion rate of creatinin was reduced to approximately 50% and 15-20% by a single-layered and by a quintuplet-layered cell sheet, respectively. On the other hand, the diffusion rate of stromal cell-derived factor 1alpha, vascular endothelial growth factor, beta2-microglobulin, and transferrin was reduced to approximately 10%, 5%, 20%, and 10%, by only a single-layered cell sheet, respectively. The diffusion of these substances were completely inhibited by a double-layered cell sheet. These results show that the permeability of substances through 3D tissue significantly decreased with the increase of the molecular weight. Therefore, the system could give a simulated living-tissue condition for measuring the permeability of substances. To our knowledge, this is the first report about measuring the permeability of substances through cell-dense 3D tissues without scaffolds. The assay system is believed to contribute to the progress of physiology, metabology, biochemistry, and pharmacokinetics. Further, the system may give some hints for developing a new dialysis membrane technology for an artificial kidney.

Recently, regenerative medicine using engineered three-dimensional (3D) tissues has been focused. In the fields of cell therapy and regenerative medicine, mesenchymal stem cells (MSCs) are attractive autologous cell sources. While, in bioengineered tissues, a 3D environment may affect the differentiation of the stem cells, little is known regarding the effect of 3D environment on cellular differentiation. In this study, MSC differentiation in in vitro 3D tissue models was assessed by human endometrial gland-derived MSCs (hEMSCs) and cell sheet technology. hEMSC sheets were layered into cell-dense 3D tissues and were cultured on porous membranes. The tissue sections revealed that chondrocyte-like cells were found within the multilayered cell sheets even at 24 h after layering. Immunostainings of chondrospecific markers were positive within those cell sheet constructs. In addition, sulfated glycosaminoglycan accumulation within the tissues increased in proportion to the numbers of layered cell sheets. The findings suggested that a high cell density and hypoxic environment in 3D tissues by layering cell sheets might accelerate a rapid differentiation of hEMSCs into chondrocytes without the help of chondro-differentiation reagents. These tissue models using cell sheets would give new insights to stem cell differentiation in 3D environment and contribute to the future application of stem cells to cartilage regenerative therapy.

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