Isolation of Exosomes from T-MSC-Conditioned Medium

To isolate exosomes, a T-MSC-conditioned medium (T-CM) was generated. Established T-MSCs [14 (link)] were grown to 80–90% confluence in 100 mm tissue culture plates, cells were washed four times with phosphate-buffered saline (PBS), and the medium was replaced with serum-free DMEM. The medium was collected after 48 h of culture, centrifuged at 1300 rpm for 5 min, and passed through a 0.2-μm filter. The CM was concentrated to 20-fold of the original concentration by centrifugal filtration (cut-off of 3K, Amicon Ultra-15, Millipore, Bedford, MA, USA). Thereafter, 1/5 volume of ExoQuick-TC reagent (System Biosciences, Palo Alto, CA, USA) was added to T-CM and mixed by vigorous inverting. After incubation at 4 °C overnight, the mixture was centrifuged at 1500× g for 30 min at 4 °C. The supernatant was removed, and final centrifugation was performed for 5 min at room temperature. The visible exosome pellet was resuspended in PBS, quantified using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and stored at −80 °C.