Isolation and Characterization of iPSC-Derived Extracellular Vesicles

The iPSCs (GD01-009) were gifted from Guidon Pharmaceutics (Beijing, China) and maintained in mTeSR1 plus (100-0276, Stemcell Technologies, Vancouver, BC, Canada) on Matrigel-coated plates (354277, Corning, NY, USA). The iPSCs were passaged as clumps using Versene Solution (15040066, Thermo Fisher Scientific, Watham, MA, USA) at a ratio of 1:4–1:6 every 4–5 days. The culture medium was substituted daily with GDEV medium (GDSJ0040, Guidon Pharmaceutics, Beijing, China) and centrifuged after daily collection to remove larger particles [42 (link)]. The supernatant was filtered and concentrated with Amicon Ultra-15 Centrifugal Filters (Ultracel-100 kDa, Merck Millipore, Burlington, MA, USA). The collected iPSC-EVs were stored at −80 °C.
The diameter, particle number and surface markers (CD9 or CD63) of iPSC-EVs were analyzed using a Flow NanoAnalyzer (NanoFCM, Xiamen, China). Transmission electron microscopy was used to identify the morphology of iPSC-EVs.