Isolation and Purification of Small Extracellular Vesicles

A classical differential UC protocol (31 (link)) was used with minor modifications to isolate sEVs. 5 × 10⁶ HPSC/HPaStec cells were seeded in a T150 cm² flasks (corning) at 50% confluence in each respective medium with 10% exosome-free FBS (Thermo Scientific, #A2720803), and the conditioned medium was harvested when cells were 70% to 80% confluent (48 h). Cell-conditioned media was cleared of cells, cell debris, and large membrane vesicles by sequential centrifugation at 500g for 30 min followed by 12,000g for an additional 30 min and was then filtered through a 0.22 μM filter unit (Millipore, #SCGP00525). This step was done to maximize quality over quantity to exclude the apoptotic bodies and other debris supported by previous publications (24 (link), 28 (link), 32 (link)).To sort by density, sEVs were collected from the cleared supernatants after centrifugation at 100,000g for 2 h in SWT32i (Beckman) swinging buckets using a Beckman Coulter ultracentrifuge (Beckman). The sEVs were washed with PBS and repurified by centrifugation at 100,000g for 2 h. The pellets were resuspended in 0.22 μM–filtered PBS. Exosomal protein quantity was estimated by Pierce bicinchoninic acid (BCA) protein assay reagent (Thermo Scientific, #23227) and Nanodrop assay (Thermo Scientific), according to the manufacturer’s instructions.