93 Massive Expansion and Cryopreservation of Beating Human iPSC-derived Cardiomyocytes 4 4. Add the required cell amount from the 1 ml cell suspension into each well of a Matrigelcoated plate with room temperature warm 2ml E8 + 5 μM Y27632 (split ratio of 1:13 is performed here). 5. Place the plate in the incubator and make side-to-side movements to equally disperse the cells in every well. 6. Each day aspirate the medium and add 2 ml of fresh room temperature hiPSC E8 complete medium per well. 7. When the hiPSCs are 80–90% confluent they are ready to start the differentiation. At this point, equal dispersed hiPSC colonies should be present in each well. CRITICAL: The starting seeding cell density is very critical for efficient cardiac differentiation. The initial plating density and/or the time of expansion prior to initiation of differentiation may require optimization for different cell lines or expansion conditions. We recommend plating at a 1:10-1:20 splitting ratio with a cell density of 80-90% within 3-4 days. Note: Karyotyping of the hiPSC lines is advised. Thereafter, hiPSC can be safely passaged for 20 times before another karyotyping is advised. Passaging over 40 times could lead in spontaneous differentiation of the hiPSC lines, resulting in inefficient differentiations. Pause point: The hiPSC can be maintained in hiPSC E8 complete medium and passaged every 3-4 days before starting differentiation and hiPSC-CM expansion. hiPSC-CM differentiation Timing: 10 min per day 8. Day 0: Prepare 3 mL/well of the appropriate concentration of CHIR per hiPSC line in CM differentiation medium. For every differentiation apply 2 concentrations of CHIR for an optimal window concentration per differentiation per plate. CRITICAL: Do not refreeze and thaw the CHIR aliquots since this molecule is unstable after thawing and freezing. Note: Other differentiation protocols can be used to generate hiPSC-CMs before the expansion. Here, we have used the B273, Heparin4 and CDM32 medium, which were all sufficient for the massive expansion of hiPSC-CMs. Note: Though we identified 7 and 8 μM CHIR in the B27 protocol as the optimal concentration for the three lines that we tested, other lines may respond to CHIR treatment differently. We recommend testing concentrations of 2-8 μM of CHIR in heparin4 or CDM32 medium and 5-10 μM of CHIR in B273 medium differentiations. 9. Day 1: On top of the original medium, add carefully 2 ml/well of 37°C-preheated CM differentiation medium. Put the plate back into the 37°C, 5% CO2, 21% O2 incubator. 10. Day 2: On top of the original medium, add carefully 1 ml/well of 37°C-preheated CM differentiation medium. Put the plate back into the 37°C, 5% CO2, 21% O2 incubator. Note: Here we use the addition of medium on day 1 and day 2 to the original medium of step 8. This protocol is effective to gradual decrease the CHIR concentration, mimicking
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