Isolation of Synovial Cells from OA Patients

This study was approved by the Medical Research Ethics Committee of Tokyo Medical and Dental University, and all study subjects provided written informed consent. Human synovium was harvested from the knees of nine donors (mean age: 76.2 ± 5.3 years) with osteoarthritis during total knee arthroplasty. The synovial tissue was digested in a solution of 3 mg/mL collagenase (Sigma-Aldrich Japan, Tokyo, Japan) at 37 °C^{10 (link)}. After 3 hours, the digested cells were filtered through a 70-μm cell strainer (Greiner Bio-One GmbH, Frickenhausen, Germany) and cultured in α-Minimal Essential Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% antibiotic-antimycotic (Thermo Fisher Scientific) and 10% fetal bovine serum (Thermo Fisher Scientific) in a humidified cell culture chamber (Tokai Hit, Fujinomiya, Japan) set at 37 °C with 5% CO₂, 20% O₂ and 75% N₂. The cells were counted with an automated cell counter (Luna-FL; Logos Biosystems, Annandale, VA, USA) in a disposable cell counting plate to determine the number of nucleated cells.