miRNA microarray analysis of murine CTL EVs separated by ion exchange method [miRNA]. miRNA microarray analysis of murine CTL EVs separated by ion exchange method [miRNA]

EVs in culture supernatant can be concentrated with removing exsomeres and 97% free proteins by MWCO 750 kDa UF. DEAE chromatography can be divided into bioactive EXO and other EVs as nucleic acid (DNA) cargo in UF-concentrated EVs. Overall design: Recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (EVs). In this study, we propose a novel method for large scale preparation of high-performance extracellular vesicles focusing on membrane negative charge. Murine cytotoxic T lymphocyte (CTL) EVs in supernatant are concentrated more than 20 times at over 97% purity without leaking by 750 kDa MWCO ultrafiltration, replaced with PBS, and subjected to ion exchange DEAE column chromatography.

细胞培养上清液中的细胞外囊泡（Extracellular Vesicles, EVs）可通过截留分子量750 kDa的超滤（Ultrafiltration, UF）技术实现浓缩，同时可去除exsomeres与97%的游离蛋白质。通过DEAE离子交换色谱，可将超滤浓缩后的EVs分为携带核酸（DNA）载荷的活性外泌体（Exosomes, EXO）与其他细胞外囊泡两类。
总体实验设计：近年来，为开展细胞外囊泡（Extracellular Vesicles, EVs）可靠生物活性研究与药物开发工作，亟需开发可替代超速离心法的新型制备方案。本研究提出一种全新的规模化制备高性能细胞外囊泡的方法，该方法聚焦于囊泡膜表面的负电荷特性。研究中，小鼠细胞毒性T淋巴细胞（Cytotoxic T Lymphocyte, CTL）上清液中的EVs经截留分子量750 kDa的超滤处理后，可实现20倍以上的浓缩，纯度达97%以上且无EVs流失；随后将浓缩产物用磷酸盐缓冲液（Phosphate Buffered Saline, PBS）进行缓冲液置换，并通过DEAE离子交换柱色谱完成分离。