用于研究和治疗应用的细胞外囊泡的纯化需要更新的方法来解决传统超速离心和其他基于尺寸的分离技术的局限性。它们的下降包括诱导的细胞外囊泡聚集,低收益率,分离过程的可扩展性和一维性差,因为细胞外囊泡的大小或沉降速度通常是唯一的选择标准。离子交换色谱法是一种有前途的替代或补充方法的候选,因为它提供了一种不同的细胞外囊泡分离方法,这是表面电荷。现在,主要是阴离子交换色谱已被评估为细胞外囊泡纯化,因为它成功地依赖于细胞外囊泡的强负表面电荷。然而,因为细胞外囊泡的结构非常复杂,阳离子交换色谱法也可以适用,由于细胞外囊泡表面上的单个阳离子结构域。这里,我们将阴离子交换色谱与不同类型的阳离子交换色谱进行了比较,以纯化也含有血浆来源杂质的血小板胞外囊泡样品。我们发现,用于阳离子交换色谱的树脂结构的选择对于结合血小板胞外囊泡至关重要,作为常规类型的阳离子交换剂,发现仅捕获和洗脱不到20%的细胞外囊泡。用触手型树脂,与阴离子交换色谱相比,阳离子交换色谱可以获得相当的血小板胞外囊泡产率(超过90%),以及卓越的纯度,特别是当它被结合到传统的阳离子交换树脂。
Purification of extracellular vesicles for research and therapeutic applications requires updated methodology to address the limitations of traditional ultracentrifugation and other size-based separation techniques. Their downfalls include induced extracellular vesicle aggregation, low yields, poor scalability and one-dimensionality of the separation process, as the size or sedimentation speed of extracellular vesicles is often the only selection criterion. Ion exchange chromatography is a promising alternative or supplementary method candidate, as it offers a different approach for extracellular vesicle separation, which is surface charge. For now, mostly anion exchange chromatography has been evaluated for extracellular vesicle purification, as it successfully relies on the strongly negative surface charge of extracellular vesicles. However, as extracellular vesicles are very complex in their structure, also cation exchange chromatography could be applicable, due to individual cationic domains on the extracellular vesicle surface. Here, we compare anion exchange chromatography to different types of cation exchange chromatography for the purification of platelet extracellular vesicle samples also containing plasma-derived impurities. We found that the choice of resin structure used for cation exchange chromatography is critical for binding platelet extracellular vesicles, as a conventional-type cation exchanger was found to only capture and elute less than 20% of extracellular vesicles. With the tentacle-type resin, it was possible to obtain comparable platelet extracellular vesicle yields (over 90%) with cation exchange chromatography compared to anion exchange chromatography, as well as superior purity, especially when it was combined to conventional cation exchange resin.