a) The double-step overlaying is schematically illustrated. Triple-layer cocultured cardiac cell sheets are overlaid on the vascular bed and perfused. After 3 days of cultivation, to provide sufficient vascularization within the first graft, the second triple-layer tissue is overlaid on the first graft and the construct is perfused for a further 3 days. Finally, the whole construct is thoroughly vascularized. ( b) The single-step overlaying with six-layer cell sheets is schematically illustrated. Six-layer cell sheet tissues are overlaid, in one step, on the vascular bed and perfused for 6 days. ( c, d) HE and Azan staining for blood-perfused double-step constructs showed that blood cells well reached the six-layer cell sheets (white arrows) and that thicker and more cell-dense tissue was successfully fabricated. ( e, f) HE and Azan staining for blood cell-perfused one-step constructs shows some necrotic cells (black arrow heads) and relatively cell-sparse tissues. ( g– i) After continuous luciferin infusion, serial bioluminescent imaging of each group was analysed. The images captured at 60 min are shown ( g, h). The intensity from the double-step procedure was significantly higher than that from the single-step procedure ( i). Data are shown as mean±s.d. (* P<0.05) ( n=4). Triple-layer cell sheets are overlaid four times on the vascular bed to produce a thick 12-layer construct ( j).