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Figure 5: (a) Schematic illustration of cell culture insert holding a multi-layered cell sheets. The insert consists of a PET frame and a porous PET membrane (the pore size: 1 mm) in the bottom. Photograph (b) represents the hematoxylin and eosin stained cross-section of quadruple-layered cell sheets after 7 days cultivation; (c), quintuple-layered cell sheets; (d), sextuple-layered cell sheets. Photograph (e) represents the section of multi-layered cell sheets stained with anti-Ki-67 antibody after 7 days cultivation on culture insert; (f), quintuple-layered cell sheets; (g), sextuple-layered cell sheets. Cross-sections were counter- stained with hematoxylin for staining nucleus. Graph (h) shows the cell viabilities of quadruple-, quintuple-, and sextuple-layered cell sheets on Day 7. The cell viabilities of multi-layered cell sheets cultured on normal cell culture dishes are the controls. Graph (i) shows the average percent of Ki-67 positive cell per 20 times magnified field in quadruple-, quintuple-, and sextuple-layered cell sheets on Day 7. Each data point represents mean SD, n = 3. ***, * versus control, p < 0.001 and p < 0.05, respectively.
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