Isolation and Characterization of Exosomes from Cultured Cells

Passage 1 SBOs were grown until 80% confluence and the medium was replaced by serum free DMEM. Conditioned medium (60 mL in total) was collected after 24 h of incubation and filtered through a 0.22 μm filter sterilizer. The collected medium was defined as nonsclerotic SBOs or sclerotic SBOs cultured conditioned medium (CM) and was stored at −80 °C before use in the subsequent experiments (Figure 1A). In this study, we used ultracentrifugation method to isolate exosomes. The SBOs CM was centrifuged using a Beckman Coulter Microfuge 18 Centrifuge (Bechman Coulter Life Sciences, Indianapolis, United States) at 300× g at 4 °C for 10 min to remove detached cells and was filtered through 0.22 μm filters (Sarstedt, Numbrecht, Germany) to remove contaminating apoptotic bodies, microvesicles, and cell debris (Figure 1B). Clarified CM was then centrifuged in a Beckman Coulter Optima^TM L-90K Ultracentrifuge at 100,000× g at 4 °C for 90 min with a Type SW41 rotor to pellet exosomes. The exosome-containing pellets were resuspended in ice-cold PBS. A second round of ultracentrifuge using Beckman Coulter OptimaTM MAX-80XP Ultracentrifuge at 100,000× g at 4 °C for 90 min with a Type TLA 110 rotor was carried out, and the final exosome pellets were resuspended in PBS after carefully removing the supernatant [22 (link)] (Figure 1B).