2.1. MSCs isolation, expansion and culture
Suprapatellar fat pad (SPFP) tissues were harvested during TKA with tissues and then used for cell isolation, and for collecting mesenchymal stem cells (MSCs) according to a previously published protocol(Katagiri et al., 2017). In brief, SPFP tissues from resections were rinsed and washed with phosphate buffer solution (PBS) (Gibco™, Life Technologies, NY, USA), and then chopped in to small pieces. After that, 0.1% collagenase type I solution (Gibco™, Life Technologies, NY, USA) was added and incubated under warm-water bath (37C) for 60 minutes then suspended with Dulbecco’s Modified Eagle Medium-high Glucose (DMEM-HG) (Gibco™, Life Technologies, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco™, Life Technologies, NY, USA ) and centrifuged at 400x for 10 minutes and the surfactant layer was removed. The resulting pellet was mixed with 15 mL of complete medium (DMEM-HG + 10% FBS + 1% L-glutamic acid (Gibco™, Life Technologies, NY, USA) + 1% Pen-strep (Gibco™, Life Technologies, NY, USA), then passed through a sterile filter/cell strainer (Corning, NY, USA) and finally seeded in T-175 tissue culture flasks (Wuxi NEST Biotechnology, Jiangsu, China) which were placed in a 37 C incubator with 5% CO2. Culture medium was replaced every 3 days. If cells expanded to more than 80% of the culture flask, then SPFP cells were detached with 0.05% Trypsin/0.1% EDTA (Gibco™, Life Technologies, NY, USA) and recultured as the first passage with complete medium through 2nd passage. Cell count and time between each passage were recorded. Suprapatellar fat pad derived mesenchymal stem cells ( SPFP-MSCs) in the first passage were trypsinized and divided for the following assays following International Society for Cellular Therapy (ISCT).