Abstract：
The development of a new large-scale purification protocol is required for research on the reliable bioactivity and drug discovery of extracellular vesicles (EVs). To address this issue, herein, we propose an effective method for preparing high-performance exosomes (EXOs) by using an anion-exchange method. Cytotoxic T-lymphocyte (CTL) EVs from 4 L of culture supernatant through a 220 nm cut-off filter are divided into two populations at a deproteinization rate of over 99.97%, which are eluted at low (0.15 M-0.3 M) and high (0.3 M-0.5 M) NaCl concentrations (approximately 2 × 1012 and 1.5 × 1012 particles, respectively) through the anion-exchange column chromatography. The former are abundant in EXO proteins, including late endosome-associated proteins and rab-family and integrin-family proteins, and functional micro (mi) RNAs, and have bioactivity for preventing tumour metastasis by depleting mesenchymal cell populations in the primary tumour lesions. By contrast, the latter is microvesicle (MV)-like particles including DNA, core histone and ribosomal proteins, and GC-rich miRNAs with unknown function, and are easily phagocytosed by mannose receptor+ Kupffer cells. Thus, the anion-exchange method is suitable for the large-scale separation of bioactive EXOs and MV-like EVs as a cargo for dangerous nucleic acids at high-purity.
摘要：
研究细胞外囊泡（EV）的可靠生物活性和药物发现需要开发新的大规模纯化方案。为了解决这个问题，在这里，我们提出了一种通过阴离子交换方法制备高性能外泌体（EXO）的有效方法。通过220nm截止过滤器从4L培养上清液中获得的细胞毒性T淋巴细胞（CTL）EV以超过99.97％的脱蛋白率分为两个群体，在低（0.15M-0.3M）和高（0.3M-0.5M）NaCl浓度（约2×1012和1.5×1012颗粒）下洗脱，分别)通过阴离子交换柱层析。前者富含EXO蛋白，包括晚期内体相关蛋白和rab家族和整合素家族蛋白，和功能性微（mi）RNA，并具有通过消耗原发性肿瘤病变中的间充质细胞群来预防肿瘤转移的生物活性。相比之下,后者是微泡（MV）样颗粒，包括DNA，核心组蛋白和核糖体蛋白，和功能未知的富含GC的miRNA，容易被甘露糖受体+枯否细胞吞噬。因此，阴离子交换法适用于大规模分离生物活性EXO和MV样EV，作为高纯度危险核酸的货物。
