Scaffold-free Cardiomyocyte Sheet Generation

DsRed-Luciferase-miPSCs were cultured in ESGRO complete PLUS Clonal Grade Medium (Merck Millipore). Cardiomyogenic differentiation was induced as previously reported without the purification process24 (link)25 (link)26 (link). Briefly, to generate embryoid bodies (EBs), 3000 cells were dissociated in each well of a round bottom plate in the presence of 0.2 μmol/L 6-bromoindirubin-3′-oxime (BIO; Merck Millipore) to activate the Wnt-signaling pathway. The effect of BIO was neutralized on day 3, and each EB was transferred to a flat bottom plate for adhesion culture on day 5. On day 14, the contracting cell clusters were dissociated, seeded on thermoresponsive dishes (5 × 10⁶ CMs/well; Upcell; CellSeed), and incubated at 37 °C for 2 days. At this time, they were then transferred to 20 °C until the cells detached spontaneously to form scaffold-free cell-sheets (Fig. 1b).