Collagenase 2

MSCs were isolated from subcutaneous adipose tissue of the adult mice (8–9 weeks old). Adipose tissue were dissected into small pieces and digested with 0.2% collagenase II (Dia-M, Russia) in a shaker-incubator at 37 °C, 120 rpm for one hour. Suspended cells were pelleted (500 g for 5 min), washed once in PBS (PanEco, Russia) and re-suspended in DMEM (PanEco, Russia) supplemented with 10% fetal bovine serum (Gibco, UK) and 2 mM l-glutamine (PanEco, Russia). MSCs were maintained at 37 °C, 5% CO₂ with culture medium being replaced every 3 days. MSCs from passages 3 and 4 were used for the experiments.
MSCs were differentiated into three lineages: adipogenic, chondrogenic and osteogenic and it was confirmed by detection of lipid droplets (Oil Red dye staining), glycosaminoglycans and mucins (1% alcian blue staining) and calcium deposits (5% AgNO₃ staining), respectively. Immune phenotype was determined using monoclonal antibodies to CD90.2-PerCP (1301575; Sony, USA), CD44-APC/Cy7 (103028; BioLegend, USA), CD73-Alexa Fluor647 (127208; BioLegend, USA), CD49e-PE (1119525; Sony, USA), Sca1-APC/Cy7(108126; BioLegend, USA), CD29-PE (102208; BioLegend, USA), CD11b-PE/Cy7 (101216; BioLegend, USA), CD10-PE (312203; BioLegend, USA), CD45-PE/Cy7 (103114; BioLegend, USA). Expression of CD markers was analyzed by flow cytometry (BD FACS Aria III (BD Bioscience, USA)).

MSCs were isolated from murine adipose tissue. The adipose tissue was incubated in 0.2% collagenase II (Dia-M, Moscow, Russia) solution for one hour in a shaker–incubator at 37 °C, 120 rpm. The cell suspension was pelleted (400× g for 5 min), washed thrice in PBS (PanEco, Moscow, Russia), and re-suspended in DMEM/F12 (PanEco, Moscow, Russia) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 2 mM L-glutamine (PanEco, Moscow, Russia). MSCs were maintained at 37 °C under 5% CO₂, with the culture medium replaced every three days. Passage 3 murine MSCs were used to isolate EVs and CIMVs. The MSCs’ immunophenotype was determined using monoclonal antibodies to Sca-1–APC/Cy7 (108126; BioLegend, San Diego, CA, USA), CD49e–PE (1119525, Sony, New York, NY, USA), and CD44–APC/Cy7 (103028, BioLegend, USA). The expression of CD markers was analyzed by flow cytometry BD FACS Aria III (BD Bioscience, San Jose, CA, USA). The cells were found to express the following immunophenotype: Sca-1+, CD49e+, CD44+, which is characteristic of mouse MSCs, and CD45- as a negative control.