Isolation and Characterization of Extracellular Vesicles from Cardiac Progenitor Cells

Human iPSC cell line ACS-1021 (ATCC, USA) and CPCs induced by ISX-9 were cultured as described [15 (link)]. In some cases, EBs and commercial human CPCs were also cultured. Conditioned media was collected and EVs were isolated by centrifugation at 3000 rpm for 30 min to remove cells and debris, followed by filtration through a 0.22 μm filter to remove the remaining debris. Then, the medium was further concentrated to 500 μl using Amicon Ultra-15 100 kDa centrifugal filter units (Millipore). Isolation of EV in the concentrated medium was carried out through qEV size exclusion columns (Izon Science). EV fractions were collected and concentrated by Amicon Ultra-4 10 kDa centrifugal filter units to a final volume of <100 μl. The purified EVs were stored at -80°C and subsequently characterized by particle size, EV markers, and electron microscopy.