96 Chapter 4 P1 P2 P3 P4 P5 1 10 100 1000 10000 Total Cell number (106) B27 CDM3 Heparin A B C B27 Heparin CDM3 P0 D11 P1 P2 P3 P4 P5 B27 Heparin CDM3 Figure 1: Massive expansion of hiPSC-CMs from various differentiation methods. (A) Schematic representation of Wnt-based directed cardiac differentiation and subsequent expansion of hiPSC-CMs generated by three different medium compositions ((B27; Lian et al., 2012; Heparin; Lin et al., 2017, and CDM3; Burridge et al., 2014). Timeline indicating B27-medium with insulin and additional CHIR99021 (CHIR) usage required for expansion. Abbreviations: CM; CM culture medium, PM; CM purification medium, RM; CM replating medium, EM; CM expansion medium, SM; CM splitting medium, P; passage. (B) Graph displaying the hiPSC-CM expansion capacity from hiPSC-CMs generated by the indicated differentiation method. One hiPSC line was used for these experiments. n=7, n=2, n=2 respectively from P0 day 11 to P5. Data are represented as mean ± SD. (C) Bright-field images at different time points of hiPSC-CM expansion with the input of the indicated differentiation methods. Scale bar, 200 μm. 28. Transfer the cell suspension to a new 15 ml tube. 29. Centrifuge the cell suspension at 200xg for 3 min at room temperature. 30. Aspirate the supernatant and resuspend the cell suspension in 1 ml/tube 37°C-preheated of the CM splitting medium supplemented with optimal CHIR concentration. 31. Count the cell suspension. Note: If cell numbers are high per tube, resuspend hiPSC-CMs in a larger amount of medium before counting the cell suspension (1 ml per 10 million). 32. Add 37°C-preheated CM splitting medium to the hiPSC-CMs, in the density and volume according to table 1.
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