Tryple 10x

Scaffolds were seeded as previously described (Wanjare et al., 2017 (link)). In brief, scaffolds (6-mm in diameter) were seeded into one face of the scaffold, while 12-mm scaffolds were seeded on both sides of the scaffolds. Singular cultures of iCMs were generated by dissociating iCMs from tissue culture dishes after differentiation using TrypLE 10X (ThermoFisher) (Gibco) and then seeding into the randomly oriented or aligned scaffold groups at a density of 1 × 10⁶ cells per scaffold in iCM culture medium consisting of RPMI 1640 containing B27 and insulin. Singular cultures of iECs were generated by dissociating iECs from tissue culture dishes after differentiation using HyQtase and seeding of 4 × 10⁴ cells onto scaffolds. Co-culture treatment groups were seeded with both iCMs and iECs, in which the cells were seeded in a sequential fashion. First, iECs were dissociated from cell culture dishes using HyQtase and seeded onto the scaffolds at a density of 4 × 10⁴ per scaffold in EGM-2MV endothelial expansion media. After 3 days, iCMs were then seeded onto either randomly oriented or aligned scaffolds at a density of 10⁶ cells per scaffold in iCM culture medium. The cell-seeded scaffolds were harvested 2 days later for in vivo transplantation.

Parental iPSCs and iPSCs harboring the homozygous P633L or R634Q mutation in RBM20 were previously generated and characterized^{24 (link)}. Cells were maintained on vitronectin (A31804, ThermoFisher) coated plates with Essential 8™ Flex (A2858501, ThermoFisher) medium and passaged with Versene (15040066, ThermoFisher). Cardiomyocyte differentiation was initiated by addition of 8 μM CHIR99021 (72054, STEMCELL Technologies) in RPMI-1640 medium supplemented with B27 without Insulin (RPMI-Insulin, A1895601, ThermoFisher). After 24 h, 1 volume of RPMI-Insulin was added and after 72 h, medium was changed to RPMI-Insulin with 2 μM Wnt-C59 (5148, Tocris). At day 5 and 7, medium was changed to RPMI-Insulin and at day 9 to RPMI with full B27 supplement (RPMI+Insulin, 17504044, ThermoFisher). At day 11, medium was changed to RPMI+Insulin without glucose and addition of 5 mM DL-lactate. At day 14, RPMI+Insulin was added and at day 16, cells were passaged with TrypLE10x (A1217701, ThermoFisher) and RPMI+Insulin supplemented with 10% knock-out serum replacement (10828028, ThermoFisher) and 1.66 μM Thiazovivin (72252, StemCell Technologies). One day after passaging, the medium was changed to RPMI+Insulin with subsequent medium exchange every 3 days. Passaging was done every 2–3 weeks.