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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Placental hypoxia and elevated levels of circulating soluble Fms-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic factor, are closely related to the pathogenesis of preeclampsia. Although sFlt-1 secretion from the placental trophoblasts is increased under hypoxic conditions, the underlying molecular mechanism remains unclear. Previously, an authentic hypoxia response element in the Flt-1 gene promoter was shown to be a potential binding site for hypoxia-inducible factors (HIFs). Here, we investigated the roles of HIF-1α and HIF-2α in Flt-1 gene expression in trophoblast-derived choriocarcinoma cell lines and cytotrophoblasts exposed to hypoxic conditions. In the cell lines, increased expression of sFlt-1 splice variants and nuclear accumulation of HIF-1α and HIF-2α were observed after hypoxic stimulation. A specific small interfering RNA or an inhibitor molecule targeting HIF-2α decreased hypoxia-induced up-regulation of Flt-1 gene expression. Moreover, in cytotrophoblasts, increased sFlt-1 mRNA expression and elevated sFlt-1 production were induced by hypoxic stimulation. Notably, hypoxia-induced elevation of sFlt-1 secretion from the cytotrophoblasts was inhibited by silencing the HIF-2α, but not HIF-1α mRNA. These findings suggest that hypoxia-induced activation of HIF-2α is essential for the increased production of sFlt-1 proteins in trophoblasts. Targeting the HIF-2α may be a novel strategy for the treatment of preeclampsia.