Abstract Extracellular vesicles that are derived from stem cells play an important role in the treatment of disease. To obtain high‐quality small extracellular vesicles (sEVs), we optimized the culture conditions of human induced pluripotent stem cells (hiPSCs), the supernatant collection time, and sEVs extraction methods. Firstly, hiPSCs were cultured in extracellular vesicles‐production medium (EVs‐PM) containing different concentrations (0%, 0.25%, 0.5%, 2%, 5%, and 20%) of extracellular vesicle‐depleted knockout serum replacement (ED‐KSR), and the culture supernatants were collected continuously for 5 days. Then, the sEVs were isolated, followed by an evaluation of their characteristics. The survival rates of the hiPSCs lines that were cultured in EVs‐PM containing 0.5% to 20% ED‐KSR were not significantly different (P > 0.05). The survival rates of the hiPSCs in 0.5% ED‐KSR after the culture supernatants were continuously collected for day 1, day 3, and day 5 were not statistically significant (P > 0.05). After 5 days of continuous collection of the supernatant, the hiPSCs expressed some pluripotent markers, while SSEA4 and TRA‐1‐60 expression changed gradually. The sEVs that were extracted by the two methods were all 50–200 nm, double‐layered and oval or round cellular vesicles and expressed the marker proteins CD63, TSG101, and HSP70. The characteristics of sEVs extracted on day 1, day 3, and day 5 were almost identical on morphology, size and the relative quantity of annexin V‐positive subpopulations. The PKH67 staining showed that the sEVs could be endocytosed by HepG2 cells and aggregated in the cytoplasm. The proliferation experiments showed that the sEVs can promote cell proliferation. In Conclusion, the 0.5% ED‐KSR is the optimal concentration, and that the hiPSCs culture supernatant can be continuously collected for 5 days while maintaining high cell viability and some pluripotent characteristics. Both of the methods extraction can be used to obtain biologically active sEVs.
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