Synovial Cell Culture Protocol

The synovial cells were cultured following previously established protocols.³⁶ (link) The synovium was digested using a solution of 3 mg/mL collagenase (Sigma–Aldrich Co., LLC, Merck KGaA, Darmstadt, Germany) at 37°C. After 3 h, the digested cells were filtered through a 70 μm cell strainer (Greiner Bio-One GmbH, Frickenhausen, Germany) and washed with phosphate-buffered saline (Thermo Fisher Scientific, MA, USA). The cells were cultured in a 10 cm dish (Nunc, Thermo Fisher Scientific) using α-minimum essential medium (α-MEM; Thermo Fisher Scientific) supplemented with 1% antibiotic–antimycotic (Thermo Fisher Scientific) and 10% fetal bovine serum (Thermo Fisher Scientific) in a cell culture incubator (Astec Co., Ltd., Fukuoka, Japan) at 37°C in a 5% CO₂ atmosphere. To evaluate the culture vessel, cells were seeded in six-well plates (BD Falcon, NJ, USA), a dish with a 15 cm diameter (Corning by Thermo Fisher Scientific), T75 flask (Sumitomo Bakelite Co., Ltd., Tokyo, Japan), and T225 flask (Sumitomo Bakelite Co.). Each cell was ruled out for mycoplasma infection by PCR.