Blood vessel formation and tissue viability were evaluated among the four groups, with and without EC coculture, and with or without FGF-2 administration in the perfusion medium. Each group was indicated as EC (−) FGF (−), EC (−) FGF (+), EC (+) FGF (−) and EC (+) FGF (+). (
a– d) HE staining after a 3-day perfusion culture. Neither a luminal structure nor perfused black ink were detected in either the EC (−) FGF (−) group or in the EC (−) FGF (+) group. In the EC (+) FGF (−) group, a luminal structure was observed, but the black ink was hardly detected within it. In the EC (+) FGF (+) group, the infused black ink thoroughly reached the blood vessel structure within cell sheet layers (red arrows). ( e– h) CD31 staining for ECs. No blood vessel structure was observed within the cell sheet layers in either the EC (−) FGF (−) group or the EC (−) FGF (+) group. Significant tubular blood vessel structures (white arrows) were observed in the EC (+) FGF (−) group and in the EC (+) FGF (+) group. cell sheet (CS) indicates cell sheet constructs and vascular bed (VB) indicates vascular beds. ( i– o) Luciferase-expressing triple-layer cell sheets were overlaid on the vascular beds. After continuous luciferin infusion, serial bioluminescent imaging of each group was analysed. The images captured at 45 min are shown ( i– l). Representative dynamic tracing is demonstrated ( m). The bioluminescent intensity at 45 and 90 min is shown ( n, o). The intensity increased in the early phase in the EC (+) FGF (−) group and the EC (+) FGF (+) group. On the other hand, it increased in the late phase in the EC (−) FGF (−) group and the EC (−) FGF (+) group. FGF-2 accelerated the increasing rate, regardless of EC coculture. Data are shown as mean±s.d. (* P<0.05) ( n=4). ( p) Sarcomeric α-actinin staining (red) with nuclei staining (blue) revealed well-differentiated sarcomeres within the cell sheet construct in the EC (+) FGF (+) group. ( q) HE staining showed that perfused black ink is occasionally detected EC(+) FGF(−) group.