94 Chapter 4 the first steps of embryonic heart development. However, other differentiation protocols such as 2 days of CHIR medium is also possible to generate hiPSC-CMs for the CM expansion. 11. Day 3: Aspirate the old medium and add carefully 3 mL/well of 37°C-preheated CM differentiation medium with 2 μM Wnt-C59 to each well of the 6-well plate. Put the plate back into the 37°C, 5% CO2, 21% O2 incubator. Note: During differentiation, the cell death rate is high. Remove dead cell debris as much as possible by shaking and aspirating the medium completely. 12. Day 5: Aspirate the medium and add 3 mL/well of 37°C-preheated CM differentiation medium. Put the plate back into the 37°C, 5% CO2, 21% O2 incubator. 13. Day 7: Aspirate the medium and add 3 mL/well of 37°C-preheated CM culture medium. Put the plate back into the 37°C, 5% CO2, 21% O2 incubator. Robust spontaneous contraction should occur by day 8-9. 14. Day 9: Aspirate the medium and add 2 mL ml/well of 37°C-preheated CM purification medium. Put the plate back into the 37°C, 5% CO2, 21% O2 incubator. 15. Day 11: The cardiomyocytes are now ready for cardiac expansion. Replating of differentiated hiPSC-CMs Timing: 30 min Between day 10 and day 14 the hiPSC-derived cardiomyocytes can be used for most successful cardiac expansion. Here, we use monolayers with spontaneous contracting cells on day 11 (Movie 1). From a 6 well plate format, select the most optimal well(s) by microscopic observe the amount of beating cardiomyocytes. CRITICAL: For efficient hiPSC-CM expansion, use wells where a beating percentage between 50-90% can be observed under the bright field microscope (confluent contracting wells roughly contain ~90% CMs). 16. Aspirate the medium and incubate hiPSC-CMs with pure TrypLE™ Select (10X) for 15-45 min at 37°C (0,75 mL of TrypLE per 9.6-cm2 growth surface). Note: Use the pure TrypLE™ Select (10X) for efficient dissociation of the hiPSC-CMs. Other reagents such as TrypLE™ Express or Trypsin will result in an inefficient dissociation and low viability. 17. After incubation for ~15 minutes, gently shake to detach the hiPSC-CMs. If cells do not detach yet, then repeat incubation for 1 or 2 times (up to 45 minutes). When cells come off the culture plate, add 2 ml per 25-cm2 warm RPMI-1640 per flask and dispense over the surface of the plate until all the cells are detached and gently mix 2-5 times using the 5 ml pipette. Note: Premature dissociation by pipetting results in shear stress-induced cell death, therefore shaking and longer incubations with TrypLE™ Select (10X) is advised. 18. Transfer the cell suspension to a 15-ml tube. 19. Centrifuge the cell suspension at 200xg for 3 min at room temperature.
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