Cardiac Myocytes from Human iPSCs

Commercially available human male and female iPSC-derived cardiac myocytes (iPS-CMs, Axol Bioscience, UK) were prepared according to the manufacturer’s instructions. Briefly, cardiac myocytes (1 ml, stored frozen in liquid N₂) thawed in a 37 °C water bath were initially suspended in 1 ml Axol Complete Cardiomyocyte Medium (warmed to 37 °C). An additional 8 ml of medium was added and mixed by gentle inversion. Cardiac myocytes were seeded (25,000 cells/well) in 1.5 ml medium in 12 well culture plates that contained glass coverslips (15-mm diameter, Warner Instruments, CT) pre-coated with Matrigel™ (Corning Life Sciences, MA). Cells were incubated for 24 h at 37 °C, 7% CO₂. Non-adherent cells were removed by rinsing with media. After 7 days in culture, 17-β-estradiol (Sigma) was added to the media at a final concentration of 1 nM. The iPS-CMs were kept in 17-β-estradiol for 24–48 h before measuring I_Ca,L or I_NCX by voltage-clamp technique. Control cells were treated with media containing DMSO (< 0.1%). Cultured iPS-CMs had a tendency to mature and exhibit spontaneous contractions by the 7th day of incubation. The male and female iPS-CMs used in this study had common features found in all iPS-CMs: spontaneous automaticity and contractions and stable ionic currents for weeks, as previously reported [34 (link)].