Dmem 10 fbs

Waste material of five female donors (median 57 yo) undergoing elective plastic surgery (liposuction) was used to obtain adipose tissue samples that were digested for 30 min at 37 °C with 0.075% w/v type I collagenase (Worthington Biochemical Co, Lakewood, NJ, USA). After digestion, samples were filtered through a cell strainer and centrifuged (1000× g, 5 min) [19 (link)]. Cells were seeded at 5 × 10³ cells/cm² in DMEM + 10% FBS (GE Healthcare, Piscataway, NJ, USA), L-glutamine and Penicillin-Streptomycin (Life Technology, Carlsbad, CA, USA). Cells were kept at 37 °C, 5% CO₂, and 95% humidity. Experiments were performed with cells at passages 4 for flow cytometry and passage 5 for EVs collection. To prime ASCs with inflammation, 10 ng/mL IFNγ was added for 48 h.

Adipose waste material from four female donors (median 54 years old, min 45, max 61) undergoing liposuction was digested with 0.075% w/v type I collagenase (30 min at 37 °C) (Worthington Biochemical Co, Lakewood, NJ, USA). Digested samples were filtered through a cell strainer and centrifuged (1000×g, 5 min). Pelleted cells were seeded at 5 × 10³ cells/cm² in DMEM + 10% FBS (GE Healthcare, Piscataway, NJ, USA) and penicillin-streptomycin (Life Technology, Carlsbad, CA, USA). Cells were cultured at 37 °C, 5% CO₂, and 95% humidity. Cells were cultured until 90% confluence with medium change each 3 days. At 90% density, cells were detached and either frozen or seeded at 4000 cells/cm². Cells were used for experiments at passage 5 at 90% confluence, 1 day after the last medium change. ASCs were primed with 10 ng/ml IFNγ for 48 h (iASCs). Afterwards, culture flasks were washed five times with PBS and medium without serum added. After 48 h, conditioned medium (secretome) was collected and further processed.

Waste adipose tissue was collected from three healthy female donors (50 ± 7 yo) undergoing liposuction and processed as already reported [60 (link)]. Briefly, tissues were digested in type I collagenase (0.075% w/v, 37 °C, 30 min) (Worthington Biochemical Co, Lakewood, NJ, USA) and filtered with a 100 μm cell strainer. After centrifugation (376× g, 5 min), pellets were suspended in DMEM + 10% FBS (GE Healthcare, Piscataway, NJ, USA) and, after count, cells seeded at 5 × 10³/cm². Cells were cultured at 37 °C, 5% CO₂ and 95% humidity and, at passage 5, analyzed by flow cytometry or seeded on the different surfaces.