Objective To compare the efficiency of four methods for extracting extracellular vesicles (EVs) from human umbilical cord mesenchymal stem cells(hUCMSCs). Methods EVs were isolated from the conditioned medium of hUCMSCs by ultracentrifugation (group A), or ultrafiltration combined with ultracentrifugation (group B), or ultrafiltration combined with polyethylene glycol precipitation (group C), or ultrafiltration combined with aqueous two phase system (group D). The total protein concentration of EVs in each group was determined by BCA method. The expression of Alix, CD9, and calnexin were detected by Western blotting. The morphology of EVs was analyzed by transmission electron microscopy. The particle size distribution and particle concentration of EVs were measured by nanoparticle tracking analysis. Results The total protein concentrations of EVs extracted by the above four methods were (1.92±1.77) μg/μL, (18.1±1.07) μg/μL, (6.33±1.02) μg/μL, (36.48±23.13) μg/μL from group A to D respectively. We observed the expression of CD9 and Alix, but not calnexin, in EVs from group A, B and C. However, the expression levels of CD9 and Alix were lowest in group C. In addition, the expression of CD9, Alix and calnexin were undetectable in EVs from group D. The particle concentrations of EVs in group A, B and C were 0.85×1011 particles/mL, 0.63×1011 particles/mL, 1.83×1011 particles/mL, respectively. Meanwhile, the particle distributions were all within the size range of EVs. We also observed the typical saucer-like membrane structure in EVs from group A, B and C. Conclusion The method of ultrafiltration combined with ultracentrifugation could be applied to the experiments demanding large amounts of EVs. The method of ultracentrifugation is recommended for the extraction of little amounts of EVs due to the lower risk of EV fragmentation.

中文翻译：

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四种提取人脐带间充质干细胞胞外囊泡的方法比较[J].

目的比较四种方法从人脐带间充质干细胞（hUCMSCs）中提取细胞外囊泡（EVs）的效率。方法采用超速离心（A组）、超滤联合超速离心（B组）、超滤联合聚乙二醇沉淀（C组）、超滤联合水相两相系统（D组）从hUCMSCs条件培养基中分离EVs ）。BCA法测定各组EVs总蛋白浓度。通过Western印迹检测Alix、CD9和calnexin的表达。通过透射电子显微镜分析EV的形态。通过纳米粒子追踪分析测量EV的粒径分布和粒子浓度。结果上述四种方法提取的EVs总蛋白浓度分别为（1.92±1.77）μg/μL、（18.1±1.07）μg/μL、（6.33±1.02）μg/μL、（36.48±23.13）μg/μL。分别为 A 至 D 组。我们在 A、B 和 C 组的 EV 中观察到 CD9 和 Alix 的表达，但没有观察到 Calnexin。然而，C 组中 CD9 和 Alix 的表达水平最低。此外，CD9、Alix 和 calnexin 的表达是在 D 组的 EV 中检测不到。 A、B 和 C 组中 EV 的颗粒浓度分别为 0.85×1011 颗粒/mL、0.63×1011 颗粒/mL、1.83×1011 颗粒/mL。同时，粒子分布均在 EV 的尺寸范围内。我们还观察到 A、B 和 C 组 EV 中典型的碟状膜结构。结论超滤结合超速离心的方法可适用于需要大量EVs的实验。由于 EV 碎裂的风险较低, 建议使用超速离心法提取少量 EV。