Figure 1.

Hypothermic storage of hiPSC-CMs as two-dimensional monolayers. hiPSC-CMs were stored for 3, 5, and 7 days at 4°C in HypoThermosol solution. Cell recovery was evaluated for 7 days after storage. (A): Evaluation of cell recovery after the storage interval and 7 days of culture using the Trypan Blue exclusion method. (B): Assessment of metabolic activity recovery using the PrestoBlue assay. The cell concentration (A) and the fluorescence values of PrestoBlue (B) are presented as a percentage in relation to the values obtained in the cells immediately before cold storage. (C): Upper panel: Viability analysis of hiPSC-CMs from the S7 condition, assessed at days 1 and 7 after storage, using the FDA (live cells; green) and PI (dead cells; red). Lower panel: Detection of caspase-3 activity using NucView Caspase-3 Substrate (red) and the MitoView mitochondrial dye (cyan) at days 1 and 7 after storage in S7 cultures. Scale bars = 100 μm. (D): Cell lysis measured by the LDH activity in the culture supernatant. Values are normalized by the number of cells. (E): Fold increase in the activity of caspases-3/7, -6, -8, -9, and -10 immediately and 24 hours after hypothermic storage in the S7 condition in relation to the cells before storage. Data are presented as mean ± SD of three (A), four (E), and five (B, D) measurements. ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001, determined by one-way analysis of variance and unpaired t test. Abbreviations: d0, 0 days after storage; d1, 1 day after storage; FDA, fluorescein diacetate; hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocyte; LDH, lactate dehydrogenase; ns, not significant; PI, propidium iodide; S3, stored for 3 days; S5, stored for 5 days; S7, stored for 7 days.