100 μm nylon strainer

Infrapatellar fat pad tissues were obtained from three female patients aged 62, 74, and 77 years undergoing TKA; these patients provided informed consent to participate. Patients with infection, rheumatic arthritis, and age older than 80 years were excluded. Tissues from patients were managed individually. The tissue was washed three times in phosphate-buffered saline, minced, and digested for 45 minutes to 1 hour at 37°C with 0.075% Type XI collagenase (Sigma) in Dulbecco modified Eagle medium (Gibco). Dulbecco modified Eagle medium containing 10% fetal bovine serum ([FBS] Gibco) was introduced to the digested solution to neutralize the enzyme activity. The cell fraction was collected by centrifuging the solution at 500 g for 10 minutes. The cell pellet was resuspended in complete culture medium (α-minimum essential medium; Gibco), 17% FBS (Gibco), 100 units/mL of penicillin (Gibco), 100 μg/mL of streptomycin (Gibco), and 2 mM of L-glutamine [Gibco]) and filtered through a 100-μm nylon strainer (Falcon) to remove large cell clusters. The cells were cultured in complete culture medium at 37°C in 5% CO 2 . After 24 hours, the medium was changed to remove nonadherent cells. Adherent cells were expanded via serial passaging. Cells from three patients at Passage 3 were stored in liquid nitrogen as a cell bank for characterization and MSC-extracellular vesicle production.