Abstract: Objective To explore the optimal method for separating and purifying small extracellular vesicles from the supernatant of mesenchymal stem cells. Methods Three standard extracellular vesicle separation procedures, including differential (ultra) centrifugation (dUC), polyethylene glycol (PEG) precipitation and QIAGEN kit method, were compared; small extracellular vesicles were extracted through ultrafiltration concentration and filtration with a 0.22 μm or 0.45 μm filter membrane, respectively, based on the standard procedure. The quality of the extracted extracellular vesicles and the efficiency of the separation methods were analyzed by comparing the operation duration and simplicity of the separation methods, and evaluating the morphological structure, particle size distribution and marker protein expression of small extracellular vesicles through transmission electron microscopy, nanoparticle tracking analysis technology(NTA)  and  Western  blot, respectively. The CCK⁃8 assay and Transwell migration assay were used to evaluate the effects of small extracellular vesicles extracted by the modified methods on the proliferation and migration of breast cancer cells. Results Small extracellular vesicles could be isolated by all these three methods, and the QIAGEN kit method has the shortest operation duration, 48 min on average, and up to 93 min after ultrafiltration concentration (P<0.0001). The PEG precipitation method had the longest operation duration, 487 min on average, and up to 547 minutes with additional ultrafiltration concentration step (P<0.0001). The average operation duration of the dUC method was 217 minutes; after the addition of the ultrafiltration conentration step, it was up to 274 minutes (P<0.0001). The typical "saucer" structure was observed in all samples under transmission electron microscopy, and the particle sizes of each sample ranged between 30 and 200 nm except for that in the QIAGEN kit method, which was above 200 nm. Among them, the dUC+0.45 μm filter membrane group had the largest number and the most complete small exbracellular vesicles structures in one field of view. Western blot results showed that positive marker proteins CD9, CD63 and TSG101 were expressed in the samples extracted by different methods, but the negative marker calnexin was not expressed. However, after 0.22 μm filter membrane filtration, the bands of small extracellular vesicle marker proteins became shallow. The NTA results showed that the proportion of small extracellular vesicles filtered by dUC+0.45 μm filter membrane was the highest, reaching 94.86%. The particle size distribution maps of samples extracted by different methods showed that the NTA results of dUC standard and the dUC+0.45 μm filter membrane groups showed a unimodal and smooth curve. Samples filtered by dUC+0.45 μm filter membrane were selected for breast cancer cell phenotype experiment, and the results showed that cell proliferation and migration were enhanced (all P<0.05). Conclusions dUC is an effective method for separating small extracellular vesicles from mesenchymal stem cells. The quality of small extracellular vesicles can be improved by filtering the cell supernatant with 0.45 μm filter membrane before ultracentrifugation. 

Key words: Tumor microenvironment, Mesenchymal stem cells, Small extracellular vesicles, Separation and purification