Pen strepto

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.

Adipose-derived MSCs (ASCs) were obtained as previously described [24 ]. Briefly, the adipose tissue was enzymatically digested (37 °C, 30 min) by 0.075% w/v type I collagenase (Worthington Biochemical Co., Lakewood, NJ, USA). After digestion, samples were filtered through a cell strainer (100 μm) and centrifuged (1000×g, 5 min). Released cells were seeded at 5 × 10³ cells/cm² in DMEM supplemented with 10% FBS (GE Healthcare, Piscataway, NJ, USA) and pen/strepto (Life Technology, Carlsbad, CA, USA). Synovial membranes were minced in small pieces and enzymatically digested (37 °C, 3 h) by 0.25% w/v type I collagenase (Worthington Biochemical Co., Freehold, NJ, USA). After digestion, samples were filtered through a cell strainer (100 μm) and centrifuged (376×g, 5 min). Cells were seeded at 5000 cell/cm² density and fibroblast-like synoviocytes (FLSs) selected for plastic adherence [25 ]. ASCs and FLSs were cultured in DMEM supplemented with 10% FBS and pen/strepto. All cell types were maintained in an incubator at 37 °C in a humidified atmosphere with 5% CO₂ and used for the following experiments between passages 3 and 5.