He Jing, Deng Taoran, Li Changyong, Li Yufeng, Wu Dongcheng, Wu Mingfu
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430072, China.
Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430075, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2020 Oct;36(10):897-902.
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 0.85×10 particles/mL, 0.63×10 particles/mL, 1.83×10 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.
目的 比较从人脐带间充质干细胞（hUCMSCs）中提取细胞外囊泡（EVs）的四种方法的效率。方法 通过超速离心（A组）、超滤联合超速离心（B组）、超滤联合聚乙二醇沉淀（C组）、超滤联合双水相系统（D组）从hUCMSCs的条件培养基中分离EVs。采用BCA法测定各组EVs的总蛋白浓度。通过蛋白质免疫印迹法检测Alix、CD9和钙连接蛋白的表达。通过透射电子显微镜分析EVs的形态。采用纳米颗粒跟踪分析测定EVs的粒径分布和颗粒浓度。结果 上述四种方法提取的EVs的总蛋白浓度从A组到D组分别为（1.92±1.77）μg/μL、（18.1±1.07）μg/μL、（6.33±1.02）μg/μL、（36.48±23.13）μg/μL。我们在A组、B组和C组的EVs中观察到CD9和Alix的表达，但未观察到钙连接蛋白的表达。然而，C组中CD9和Alix的表达水平最低。此外，在D组的EVs中未检测到CD9、Alix和钙连接蛋白的表达。A组、B组和C组中EVs的颗粒浓度分别为0.85×10颗粒/mL、0.63×10颗粒/mL、1.83×10颗粒/mL。同时，颗粒分布均在EVs的大小范围内。我们还在A组、B组和C组的EVs中观察到典型的碟状膜结构。结论 超滤联合超速离心法可应用于需要大量EVs的实验。由于EVs破碎风险较低，超速离心法推荐用于少量EVs的提取。
