Staubach Simon, Tertel Tobias, Walkenfort Bernd, Buschmann Dominik, Pfaffl Michael W, Weber Gerhard, Giebel Bernd
Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen 45147, Germany.
Imaging Center Essen (IMCES) Electron Microscopy Unit, University Hospital Essen, University of Duisburg-Essen, Essen 45147, Germany.
Extracell Vesicles Circ Nucl Acids. 2022 Mar 16;3(1):31-48. doi: 10.20517/evcna.2021.26. eCollection 2022.
Despite intensive research during the last decade, it remains challenging to prepare extracellular vesicles (EVs) of high purity, especially from primary body liquids or protein-rich conditioned media. For now, time-consuming combinations of at least two orthogonal methods, e.g., density and size separation, are required to enrich EVs to high purity, often at the expense of processing time. Therefore, novel technologies are required that allow EV preparation in acceptable time intervals and to fair purities. Free-flow electrophoresis (FFE) constitutes a well-established semi-preparative method to separate and prepare analytes, e.g., by inherent differences in their electric charges. FFE combines a flow-driven longitudinal transport of sample material with vertical electrophoresis and allows the separation of sample components into up to 96 different fractions. It was our aim to evaluate the potential of FFE for the separation of EVs from other sample components of EV-containing protein-rich conditioned cell culture media.
Exemplarily, conditioned media of mesenchymal stem/stromal cells raised in the presence of EV-containing 10% human platelet lysate were processed. We analyzed the obtained fractions by different technologies, including imaging flow cytometry, western blot and nanoparticle tracking analysis.
We demonstrate that FFE quickly and reproducibly separates EVs from a huge proportion of molecules included in the original sample.
Our results qualify FFE as a feasible, quick and reproducible technology for the preparation of EVs.
尽管在过去十年中进行了深入研究，但制备高纯度的细胞外囊泡（EVs）仍然具有挑战性，尤其是从原代体液或富含蛋白质的条件培养基中获取时。目前，需要至少两种正交方法（如密度和尺寸分离）的耗时组合来将EVs富集至高纯度，这往往是以牺牲处理时间为代价的。因此，需要新技术能够在可接受的时间间隔内制备出纯度合适的EVs。自由流电泳（FFE）是一种成熟的半制备方法，用于分离和制备分析物，例如根据其电荷的固有差异进行分离。FFE将样品材料的流动驱动纵向传输与垂直电泳相结合，可将样品成分分离成多达96个不同的馏分。我们的目的是评估FFE从富含蛋白质的含EVs条件细胞培养基的其他样品成分中分离EVs的潜力。
举例来说，对在含有10%人血小板裂解物的含EVs环境中培养的间充质干/基质细胞的条件培养基进行处理。我们通过不同技术分析获得的馏分，包括成像流式细胞术、蛋白质印迹和纳米颗粒跟踪分析。
我们证明FFE能够快速且可重复地从原始样品中包含的大量分子中分离出EVs。
我们的结果表明FFE是一种用于制备EVs的可行、快速且可重复的技术。
