Isolation and Characterization of Human Adipose-Derived Stem Cells

Human adipose tissue samples were obtained from waste material of five female donors (range 61-44 years old) who had undergone elective plastic surgery. 0.075% w/v type I collagenase (Worthington Biochemical Co, Lakewood, NJ, USA) was used to digest adipose tissue (37 °C, 30 min). After digestion, samples were filtered through a cell strainer and centrifuged (1000× g, 5 min) [56 (link)]. Cells in the pellet were seeded at 5 × 10³ cells/cm² in DMEM supplemented with 10% FBS (GE Healthcare, Piscataway, NJ, USA), L-glutamine and pen/strepto (Life Technology, Carlsbad, CA, USA). Culture were kept at 37 °C, 5% CO₂ and 95% humidity. To mimic inflammatory environment, ASCs were treated with cytokines resembling those quantified in OA synovial fluid (40 pg/mL IFNγ, 10 pg/mL Il-1β and 5 pg/mL TNFα) [57 (link)]. Cells were characterized by flow cytometry using positive or negative MSC markers (CD44, CD73, CD90 and CD105 or CD45; Miltenyi Biotec, Bergisch Gladbach, Germany) and hematopoietic negative marker (CD34) [58 (link),59 (link)] as described previously with a CytoFLEX flow cytometer (Beckman Coulter, Fullerton, CA, USA) collecting a minimum of 30,000 events [60 (link)]. Cells were used for the experiments between passage 3 and 5.