Upper chamber of transwell

For scratch closure assay, HDFs were plated at 4 × 10⁴ cells/cm² in 24-well plate, and incubated in growth medium for 24 h. After serum starvation for 24 h, the monolayer of cells was scratched with a sterile pipette tip. Subsequently, cells were incubated with iPSC-CENVs in serum-free DMEM/F12 for the indicated time, and closure of the scratched area by cell migration was monitored under an optical microscope. For transwell migration assay, HDFs were plated onto upper chamber of transwell (Corning, Lowell, MA, USA), and incubated in growth medium for 24 h. Following the treatment with iPSC-CENVs for 48 h in serum-free DMEM/F12, both cells that remained on the upper side and those that had migrated toward the opposite side of the trans-membrane were fixed by 4% paraformaldehyde. After removal of the cells on the upper side by cotton swap, the cells at the bottom side of membrane were stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, and observed under an optical microscope. For quantitative analysis, the membrane was treated with 50% acetic acid to dissolve the crystal violet, and the optical absorbance at 560 nm was measured using a microplate reader.