Department of Microbiology and Immunology, The University of North Carolina, Chapel Hill, NC, USA.
Department of Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, NC, USA.
Methods Mol Biol. 2023;2668:45-55. doi: 10.1007/978-1-0716-3203-1_5.
Extracellular vesicle (EV) isolation from conditioned cell culture medium has been a challenging topic. It is particularly difficult to obtain pure and intact EVs at a large scale. The commonly used methods such as differential centrifugation, ultracentrifugation, size exclusion chromatography, polyethylene glycol (PEG) precipitation, filtration, and affinity-based purification each have their advantages and limitations. Here, we present a tangential-flow filtration (TFF) based, multi-step purification protocol that combines filtration, PEG precipitation, and Capto Core 700 multimodal chromatography (MMC) to isolate EVs at high purity from large volumes of cell culture conditioned medium. Inserting the TFF step before PEG precipitation removes proteins, which may aggregate in subsequent steps and co-purify with EVs.
从细胞培养条件培养基中分离细胞外囊泡（EV）一直是一个具有挑战性的话题。特别是，很难在大规模下获得纯净和完整的 EV。常用的方法，如差速离心、超速离心、尺寸排阻色谱法、聚乙二醇（PEG）沉淀、过滤和基于亲和性的纯化，各有其优点和局限性。在这里，我们提出了一种基于切向流过滤（TFF）的多步纯化方案，该方案结合了过滤、PEG 沉淀和 Capto Core 700 多模式色谱（MMC），可从大量细胞培养条件培养基中以高纯度分离 EV。在 PEG 沉淀之前插入 TFF 步骤可以去除可能在后续步骤中聚集并与 EV 共同纯化的蛋白质。
