Isolation and culture of mouse adipose-derived stem cells

ADSCs were isolated from adipose tissue from C57BL/6 mice as previously described [44, 45]. In brief, adipose tissue isolated from the proximal limb was digested by 0.075% collagenase IV (Cat. 9001-12-1; Sigma, USA) for 60 min at 37° C in a mechanical horizontal rotator at a speed of 180 rpm. Collagenase IV was dissolved in complete medium containing DMEM/F12 (Cat. SH30023.01; Hyclone, USA) and 10 % fetal bovine serum (FBS; Cat. ST303302, PAN, Germany). After digestion, the samples were centrifugated at 200×g for 10 min and the cell pellets resuspended in complete medium (DMEM/F12 and 10% EV-free serum). The medium was then placed on 10 cm culture dishes in an incubator at 37° C in a 5% CO₂ atmosphere. The extraction methods were based on previous references [44, 45] and remained undisturbed for three days prior to collection of EVs. Characterization of ADSCs was performed by flow cytometry analysis of CD29, CD45, and CD90 (positive cell surface markers) and CD34, CD105, and CD106 (negative controls).