尽管对于理解细胞外囊泡（EV）在生理和疾病中的生物活性以及开发治疗应用感兴趣，但EV制备方法的影响仍然几乎未被探索。在本研究中，我们采用了密度梯度超速离心结合尺寸排阻色谱法（DG-SEC），差速超速离心法（dUC）和/或独立的SEC（sSEC）来分离不同癌细胞和/或癌相关成纤维细胞（CAF）培养的培养基。我们对富含EV但蛋白质减少和不富含EV但蛋白质增加的DG-SEC分离液，以及富含EV的dUC和sSEC制备进行质量控制，包括颗粒数量、蛋白质浓度、选择的蛋白质组成和超结构的表征，还对它们的细胞因子含量进行了表征，并通过测量表面标记物表达和/或细胞因子分泌来剂量依赖性地评估了单核细胞源性树突状细胞（MoDC）的成熟。来自不同癌细胞系或CAF培养的培养基的DG-SEC制备的EV不含可溶性免疫抑制细胞因子（如VEGF-A和MCP-1），并能高效刺激MoDC的成熟。相反，富含EV的dUC或sSEC制备物中不含这些可溶性细胞因子，并且无法使MoDC成熟。通过SEC对dUC EV制备物进行后续处理可以剂量依赖性地恢复免疫调节的生物活性。总的来说，我们的结果表明，依赖于方法的目标外源性富集可溶性细胞因子对于研究EV的免疫调节生物活性具有涉及，并需要谨慎考虑。© 2023 The Authors. Journal of Extracellular Vesicles, 由Wiley Periodicals, LLC代表国际细胞外囊泡学会出版。

Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration.© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.