Figure 4.

Characterization of hiPSC-CMs stored as three-dimensional (3D) aggregates in hypothermic conditions for up to 7 days. (A, C): After hypothermic storage and 1 week in culture after storage, cells were characterized by flow cytometry (A) and immunofluorescence staining (C) for cardiac-specific markers. (B): The regained of contractile activity was also assessed by phase-contrast microscopy. (A): Percentage of SIRPA, VCAM, and cTNT-positive cells determined by flow cytometry. (B): The beating frequency (beats per minute) after all storage intervals (S3, S5, and S7) was monitored until day 4 of culture after storage. The results were normalized to the beating frequency measured in the control cells not subjected to cold storage. (C): Immunofluorescence analysis of hiPSC-CMs (from plated 3D aggregates) after hypothermic storage for the CM-specific markers: sarcomeric α-actinin, Troponin C, Troponin I, and Titin (red) antibodies. F-actin is stained with phalloidin (green). Nuclei were counterstained with Hoechst (blue). Scale bars = 30 μm. Abbreviations: cTNT, cardiac troponin T; hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocyte; S3, stored for 3 days; S5, stored for 5 days; S7, stored for 7 days. SIRPA, signal-regulatory protein alpha; VCAM, vascular cell adhesion molecule.