98 Chapter 4 A B P1 (~ D16) P1 (~ D18) P1 (~ D19) P1 (~ D20) P1 (~ D15) P0 P1 P2 P3 P4 P5 Figure 2: Visualized timeline of hiPSC-CM expansion. (A) Timeline diagram displaying the steps required for expansion and passaging of hiPSC-CMs. (B) Time-lapse images of cell morphology at the indicated passage numbers and/or culture days (D). Left: Images of cell confluency on day of passaging for P0-5. Right: Daily consecutive images of P1 hiPSC-CMs demonstrating cell seeding density and growth speed between P1 and P2. Replating at day 151-15, start of CHIR9021 (CHIR) treatment at day 12-16 the next day, cell cluster formation at day 14-19 is observed the days after and a nearly confluent monolayer before passaging at D18-206-21 the next passage. Scale bar: 200μm. Abbreviations: D; day, P; passage. Thawing of hiPSC-derived CMs Timing: 30 min 41. Prepare 9 ml of RPMI medium in a 15-ml tube on RT. 42. Prepare a Matrigel coated T75 per 3-6 x 106 and a T175 for 7-14 x 106 hiPSC-CMs. 43. Collect the vial with hiPSC-CMs from the liquid nitrogen and thaw the cells at 37 °C till only a little clump is visible. Do not thaw more than three vials of CMs at one time.
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