Cell culture incubator

This study was approved by the Medical Research Ethics Committee of Tokyo Medical and Dental University, and informed consent was obtained from all study subjects. Human synovium was harvested from the knees of four female donors with osteoarthritis (OA) during total knee arthroplasty operations. The average age and standard deviation of the patients was 71 ± 6 years. The synovium was minced and digested in a solution of 3 mg/mL collagenase (Sigma-Aldrich Japan, Tokyo, Japan) at 37 °C for 3 h and the digested cells were filtered through a 70 μm cell strainer (Greiner Bio-One GmbH, Frickenhausen, Germany). The obtained nucleated cells were suspended in α-MEM (Thermo Fisher Scientific, Rockford IL, USA) supplemented with 1% antibiotic-antimycotic (Thermo Fisher Scientific) and 10% FBS (Thermo Fisher Scientific) and cultured in a cell culture incubator (Astec Co. Ltd., Fukuoka, Japan) in 5% CO₂ at 37 °C for 14 days. When the MSCs reached 70–80% confluence, they were replated continuously. Synovial MSCs of passage 2 were used in this study.

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.

Human synovium was harvested from the knees of 14 donors (65–80 years of age) with osteoarthritis during total knee arthroplasty. Synovium was digested in a solution of 3 mg/mL collagenase (Sigma-Aldrich Japan, Tokyo, Japan) at 37 °C. After 3 h, the digested cells were filtered through a 70-μm cell strainer (Greiner Bio-One GmbH, Frickenhausen, Germany). The cells were cultured in α-minimum essential medium (α-MEM; Thermo Fisher Scientific) supplemented with 1% antibiotic-antimycotic (Thermo Fisher Scientific) and 10% fetal bovine serum (FBS) in a cell culture incubator (Astec Co. Ltd., Fukuoka, Japan) at 37 °C under 5% CO₂. These cells were counted via an automated cell counter (Luna-FL, Logos Biosystems, VA, USA) with a disposable cell counting plate to determine the number of nucleated cells.