Figure 2.

Hypothermic storage of hiPSC-CMs as three-dimensional aggregates. Aggregates were stored for 5 and 7 days at 4°C in HypoThermosol solution. Cell recovery was evaluated for 7 days after storage. (A): Cell viability at day 1 after storage assessed by using fluorescein diacetate (live cells; green) and propidium iodide (dead cells; red) dyes. (B): Evaluation of metabolic activity recovery after storage by PrestoBlue assay. The fluorescence values are presented as a percentage in relation to the values obtained in the cells immediately before cold storage. (C): Fold increase in the activity of caspases-3/7, -6, -8, -9, and -10 immediately and 24 hours after hypothermic storage in S7 condition in relation to the cells before storage. (D, E): Scanning electron microscopy of hiPSC-CM aggregates from S7 condition after 7 days in culture after storage (D) and the control (hiPSC-CM aggregates not subjected to cold storage, i.e., maintained in culture during the storage period) (E). The boxed regions were magnified to show cell boundaries and cell-cell contacts (arrow). Scale bars = 200 μm (B), 10 μm (C left, D), and 1 μm (C middle and right). Data are presented as mean ± SD of 4 (C) and 15 (B) measurements. ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001; ns as determined by unpaired t test. Abbreviations: d0, 0 days after storage; d1, 1 day after storage; hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocyte; ns, not significant; S5, stored for 5 days; S7, stored for 7 days.