Wang Jie-Min, Li Yong-Jiang, Wu Jun-Yong, Cai Jia-Xin, Wen Jing, Xiang Da-Xiong, Hu Xiong-Bin, Li Wen-Qun
Department of Pharmacy, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
Hunan Provincial Engineering Research Centre of Translational Medicine and Innovative Drug, Changsha, Hunan, China.
Cell Biosci. 2021 Feb 10;11(1):37. doi: 10.1186/s13578-021-00550-3.
Small extracellular vesicles (sEVs) are nanosized vesicles involved in cell-to-cell communication. sEVs have been widely studied for clinical applications such as early detection of diseases and as therapeutics. Various methods for sEVs isolation are been using, but different methods may result in different qualities of sEVs and impact downstream analysis and applications. Here, we compared current isolation methods and performed a comparative analysis of sEVs from supernatant of cultured pancreatic cancer cells.
Ultracentrifugation, ultrafiltration and co-precipitation as concentration methods were firstly evaluated for yield, size, morphology and protein level of pellets. Then, isolate sEVs obtained by four different purification methods: size exclusion chromatography, density gradient ultracentrifugation, ultracentrifugation, and immunoaffinity capturing, were analysed and compared.
For the concentration process, ultracentrifugation method obtained high quality and high concentration of pellets. For the purification process, immunoaffinity capturing method obtained the purest sEVs with less contaminants, while density gradient ultracentrifugation-based method obtained sEVs with the smallest size. Proteomic analysis revealed distinct protein contents of purified sEVs from different methods.
For isolating sEVs derived from supernatant of cultured pancreatic cancer cell line, ultracentrifugation-based method is recommended for concentration of sEVs, density gradient ultracentrifugation-based method may be applied for obtaining purified sEVs with controlled size, immunoaffinity capturing may be suitable for studies requiring sEVs with high purity but may loss subtypes of sEVs without specific protein marker.
小细胞外囊泡（sEVs）是参与细胞间通讯的纳米级囊泡。sEVs已被广泛研究用于疾病早期检测和治疗等临床应用。目前使用了多种sEVs分离方法，但不同方法可能导致sEVs质量不同，并影响下游分析和应用。在此，我们比较了当前的分离方法，并对培养的胰腺癌细胞上清液中的sEVs进行了比较分析。
首先评估超速离心、超滤和共沉淀作为浓缩方法时沉淀的产量、大小、形态和蛋白质水平。然后，对通过四种不同纯化方法获得的sEVs进行分析和比较，这四种方法分别是尺寸排阻色谱法、密度梯度超速离心法、超速离心法和免疫亲和捕获法。
对于浓缩过程，超速离心法获得了高质量和高浓度的沉淀。对于纯化过程，免疫亲和捕获法获得的sEVs最纯，污染物较少，而基于密度梯度超速离心的方法获得的sEVs尺寸最小。蛋白质组学分析揭示了不同方法纯化的sEVs具有不同的蛋白质含量。
对于分离源自培养的胰腺癌细胞系上清液的sEVs，推荐使用基于超速离心的方法浓缩sEVs，基于密度梯度超速离心的方法可用于获得尺寸可控的纯化sEVs，免疫亲和捕获法可能适用于需要高纯度sEVs的研究，但可能会丢失没有特定蛋白质标记的sEVs亚型。
