110 Chapter 5 METHODS Maintenance of hiPSC. hiPSC obtained from the Stanford Cardiovascular Institute Biobank (SCVI-111 and SCVI-273) were non-enzymatically passaged using 0.5 mM EDTA (Thermo Fisher, Waltham, MA, USA) every 4 days as previously described.3 In brief, cells were incubated with 0.5 mM EDTA-PBS at room temperature for 5–8 min until cells began to separate uniformly throughout the colonies. PBS-EDTA was removed and hiPSC colonies were washed off swiftly using 1 mL E8 complete medium (Thermo Fisher, Waltham, MA, USA). hiPSC clumps were passaged in a ratio of 1:10 routinely at 80% confluence. To improve cell survival, split ratio reliability and to reduce selective pressure, 10 µM of ROCK inhibitor (Y-27632, Stemcell Technologies, Vancouver, BC, Canada) was added to the E8 complete medium for the first 24 h after cell passaging. Matrigel (Corning, Somerville, MA, USA, 1:400) coated plates (Corning, Somerville, MA, USA) were used for hiPSC culture and PSC Cryopreservtion Kit (Gibco, Waltham, MA, USA) was used for the cryopreservation of hiPSC. Differentiation of hiPSC into Cardiomyocytes. To obtain hiPSC-derived CMs, hiPSCs were grown to ~90% confluence in a 6-well format. hiPSC cells were maintained in E8 complete medium for at least three passages before starting cardiac lineage differentiations. The cells were efficiently differentiated as previously described, using the RPMI/B27 minus insulin medium (Gibco, Waltham, MA, USA) supplemented with 7–8 µM CHIR99021 (cell line specific) (SelleckChem, Houston, TX, USA) for the first 2 days and 2 µM Wnt-C59 (SelleckChem, Houston, TX, USA) for another 48 h. The proliferation of hiPSC-CMs can be conducted by the removal of cell–cell contacts and small-molecule inhibition with CHIR99021.3 Subsequential multiple low-density passaging and reintroduction of 2–3 µM CHIR99021 (cell line specific) to the RPMI/ B27 media results in an expansion of hiPSC-CMs.3 Metabolic Maturation of iPSC-CMs. In order to mature the CMs, we used a previously described metabolic maturation medium.24 After re-plating and purification, the RPMI/B27 medium was replaced by the maturation medium for a minimum of 3 weeks. Metabolic maturation medium was composed in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA, 11875119) with B27 supplement (Thermo Fisher Scientific, Waltham, MA, USA, 17504044). Maturation media was composed in DMEM without glucose (Thermo Fisher Scientific, Waltham, MA, USA, 11966025) supplemented with 3 mM glucose (Sigma Aldrich, Saint Louis, MO, USA, G7021), 10 mM L-lactate (Sigma Aldrich, Saint Louis, MO, USA, 71718), 5 μg/mL Vitamin B12 (Sigma Aldrich, Saint Luis, MO, USA, V6629), 0.82 μM Biotin (Sigma Aldrich, Saint Louis, MO, USA, B4639), 5 mM Creatine monohydrate (Sigma Aldrich, Saint Louis, MO, USA, C3630), 2 mM Taurine (Sigma Aldrich, Saint Louis, MO, USA, T0625), 2 mM L-carnitine (Sigma Aldrich, Saint Louis, MO, USA, C0283), 0.5 mM Ascorbic acid (Sigma Aldrich, Saint Louis, MO, USA, A8960), 1 × NEAA (Thermo Fisher Scientific, Waltham, MA, USA, 11140), 0.5% (w/v) Albumax (Thermo
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