103 Massive Expansion and Cryopreservation of Beating Human iPSC-derived Cardiomyocytes 4 Troubleshooting Problem 1: There are many dead hiPSC after passaging, is this normal? Potential Solution: The hiPSCs need to be monitored under a microscope and the dissociation needs to be stopped at the right time. When light sheds through the colonies and the hiPSCs can be easily flushed off the plate, the dissociation is successful. Use 0.5 mM EDTA solution and 10 μM Y27632 in the E8 complete medium is necessary. Problem 2: The hiPSC colonies are too dense in the middle, is this a problem? Potential Solution: The hiPSCs need to be equally divided over the wells to enable full monolayer differentiation with high efficiencies. Mechanically dissociate the detached hiPSC 5-10 times till the clumps are only 3-5 cells big. Move plate more times side-to-side in the incubator and leave cells for 24 hours. Problem 3: The hiPSCs look abnormal or the proliferation speed is slow. Potential Solution: Wait for some passages before starting cardiac differentiation. Perform mycoplasma test and karyotyping to rule out any hiPSC cell line abnormalities. Problem 4: No spontaneous contraction on day 11. Potential Solution: See solutions concerning problems during the cultivation of hiPSCs. Optimize the CHIR concentrations from 5-10 μM per hiPSC line. Do not re-freeze CHIR aliquots. Problem 5: Many dead hiPSC-CMs are present upon thawing, is this normal? Potential Solution: We observe a cell viability between 60-90% upon thawing by using CMs on passage 1-2. Pipette cells carefully with a 5 ml pipette. Place the thawed cells directly into the replating medium. Add 1:100 Revitacell to promote cell survival and 10% KO serum can be added to promote cell survival after thawing. Problem 6: No hiPSC-CMs are attached to the culture flask after replating. Potential Solution: Allow Matrigel to incubate at 37C for 1 hour. Check culture flask on complete coating coverage. Pipette cells carefully with a 5 ml pipette. Add 1:100 Revitacell to promote cell survival. Add 10% KO serum to promote cell survival. Seed cell number as described in Table 1. Problem 7: Many dead hiPSC-CMs after replating, is this normal? Potential Solution: Pipette carefully with 5 ml pipette, add 10 μM Y27632 to promote cell survival, add 10% KO serum to promote cell survival, decrease CHIR concentration, seed cell number as described in Table 1.
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