Isolation and Culture of Tonsil-Derived Mesenchymal Stem Cells

T-MSCs were isolated from human palatine tonsils obtained from patients undergoing tonsillectomy at Ewha Womans University Mok-Dong Hospital (Seoul, South Korea; approved by the Institutional Review Board; no. EUMC 2018-01-011-002), and were cultured in DMEM high glucose medium (Welgene, Gyeongsan, South Korea) supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin (Welgene) [20 (link),21 (link)]. Culture media was changed every 3–4 days. To collect CM, T-MSCs were cultured to 80% confluency. After replacing the culture medium with fresh serum-free medium, cells were incubated for an additional 48 h and CM was harvested. CM was concentrated using a 3-kDa Amicon Ultra centrifugal filter unit (EMD Millipore, Darmstadt, Germany) by centrifugation at 4500 rpm at 4 °C for 1 h.