In regenerative medicine, extracellular vesicles (EVs) are considered as a promising cell-free approach. EVs are lipid bilayer-enclosed vesicles secreted by cells and are key players in intercellular communication. EV-based therapeutic approaches have unique advantages over the use of cell-based therapies, such as a high biological, but low immunogenic and tumorigenic potential. To analyze the purity and biochemical composition of EV preparations, the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins coisolated/recovered with EVs. Traditional methods for EV characterization, such as Western blotting, require a relatively high EV sample/protein input for the analysis of one protein. We here evaluate a combined Western and bead-based multiplex platform, called DigiWest, for its ability to detect simultaneously multiple EV markers in an EV-containing sample with inherent low protein input. DigiWest analysis was performed on EVs from various sources and species, including mesenchymal stromal cells, notochordal cells, and milk, from human, pig, and dog. The study established a panel of nine antibodies that can be used as cross-species for the detection of general EV markers and coisolates in accordance with the ISEV guidelines. This optimized panel facilitates the parallel evaluation of EV-containing samples, allowing for a comprehensive characterization and assessment of their purity. The total protein input for marker analysis with DigiWest was 1 μg for all nine antibodies, compared with ∼10 μg protein input required for traditional Western blotting for one antibody. These findings demonstrate the potential of the DigiWest technique for characterizing various types of EVs in the regenerative medicine field.

中文翻译：

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用于表征细胞外囊泡的西方和基于微珠的多重平台相结合。

在再生医学中，细胞外囊泡（EV）被认为是一种有前途的无细胞方法。EV 是细胞分泌的脂质双层封闭的囊泡，是细胞间通讯的关键参与者。与使用细胞疗法相比，基于EV的治疗方法具有独特的优势，例如生物活性高，但免疫原性和致瘤潜力低。为了分析 EV 制剂的纯度和生化成分，国际细胞外囊泡协会 (ISEV) 制定了指南，建议分析多种 (EV) 标记物以及与 EV 共分离/回收的蛋白质。EV 表征的传统方法（例如蛋白质印迹）需要相对较高的 EV 样品/蛋白质输入来分析一种蛋白质。我们在这里评估了一个组合的蛋白质印迹和基于珠子的多重平台，称为 DigiWest，它能够同时检测含有 EV 的样品中的多个 EV 标记，并且具有固有的低蛋白质输入。DigiWest 分析对来自不同来源和物种的 EV 进行，包括来自人类、猪和狗的间充质基质细胞、脊索细胞和牛奶。该研究建立了一组由九种抗体组成的抗体组，可根据 ISEV 指南用作跨物种检测一般 EV 标记物和共分离物。这种优化的面板有助于对含有 EV 的样品进行并行评估，从而对其纯度进行全面表征和评估。对于所有九种抗体，使用 DigiWest 进行标记物分析的总蛋白质输入量均为 1 μg，而传统蛋白质印迹法中一种抗体所需的蛋白质输入量约为 10 μg。这些发现证明了 DigiWest 技术在表征再生医学领域各种类型电动汽车的潜力。