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 (37∘C) 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).