Fig. 1

Effects of vitrification on cell shape and viability. a Vitrification method. The cell-sheet construct was immersed in the protective vehicle containing ethylene glycol, sucrose, and carboxyl poly-l-lysine; wrapped in a thin film using the mesh as a substrate for the cell sheet; and frozen above liquid nitrogen to achieve vitrification. b Cell sheet shape before and after cryopreservation. A representative image of the fresh group was obtained before cryopreservation. The cryopreserved cell-sheet constructs of the vitrification group were thawed at 2 days (D), 1 week (W), 1 month (M), and 3 months for assessment. Scale bar, 1 cm. c HE staining of the cell sheet. A representative image of the fresh group was obtained before cryopreservation. The cryopreserved cell-sheet constructs of the vitrification group were thawed at 2 days, 1 week, 1 month, and 3 months for assessment. Scale bar, 100 μm. d Cell viability in the cell sheet. The fresh group was assessed before cryopreservation. The cryopreserved cell-sheet constructs of the vitrification group were thawed at 2 days, 1 week, 1 month, and 3 months to evaluate cell viability. Results are expressed as the means ± SD (n = 8 independent experiments). e Apoptotic cells in the cell sheet. Frozen sections were immunostained with Annexin 5. The ratio of Annexin 5 (+) to all cells was calculated. The fresh group was assessed before cryopreservation. The cryopreserved cell-sheet constructs of the vitrification group were thawed at 2 days, 1 week, 1 month, and 3 months to evaluate apoptosis cell numbers. Results are expressed as the means ± SD (n = 8 independent experiments); *p < 0.05