Fibrinogen

hPSC-derived cardiac cells (3TCC-hCMPs: 1.4 million hPSC-CMs, 0.2 million hPSC-AECs, and 0.2 million hPSC-SMCs; 4TCC-hCMPs: 1.4 million hPSC-CMs, 0.2 million hPSC-AECs, 0.2 million hPSC-SMCs, and 0.2 million hPSC-CFs) were suspended in 0.20 mL fibrinogen solution (0.12 mL fibrinogen, 25 mg/mL, Sigma-Aldrich; 0.02 mL growth factor reduced Matrigel, Corning; and 0.56 mL HEPES, 20 mM, pH 7.4, Corning); then, the cell-containing fibrinogen solution was mixed with thrombin solution (0.004 mL thrombin, 80 U/mL, MP Biomedicals; 0.001 mL CaCl₂, 2 M, and 0.3 mL DMEM high glucose, Gibco) in a mould (internal dimensions: 1 cm × 1 cm; height: 2 mm) that had been precoated with 5% pluronic. The mixture solidified within a few minutes, and then culture medium (10% FCS, 2% B27 plus insulin, 2 mg/mL ε-aminocaproic acid, 10 µM ROCK inhibitor in DMEM medium) was added to the mould and the dish. Twenty-four hours later, the culture medium was replaced with DMEM containing 2% FCS, 2% B27 plus insulin, and 2 mg/mL ε-aminocaproic acid, and the patches were cultured at 37°C on a rocking (45 rpm) platform for 14 days.^{14 (link),24 (link)} The culture medium was changed every 2 days, and synchronous beating of hPSC-CMs across the entire patch typically appeared on the second day after patch fabrication. hCMPs were trimmed to 0.5 cm × 0.6 cm × 2 mm before transplantation into mice.