细胞外囊泡 (EV) 作为促进细胞间通讯的化学信使。新的证据表明，肺组织来源的 EV 在肺部生理过程中发挥着关键作用，并具有作为肺部疾病的生物标志物和治疗方法的潜力。已经提出了多种方法来分离肺组织来源的 EV。然而，不同组织预处理对肺 EV 分离和随后疾病生物标志物发现的影响尚未得到全面研究。在本研究中，我们使用基于不同组织解离原理的三种方法比较了从肺组织中分离的 EV 的物理特性、回收率和蛋白质组成。从方法上讲，根据血液灌注和温和网格对 EV 产量和纯度的影响，强调了它们的有益作用。这些结果表明，不同的方法丰富了不同的 EV 亚群，这些亚群在蛋白质含量和表面特性方面表现出显着差异。这些差异直接影响与肺部疾病相关的标记蛋白的诊断检测，包括肺部肿瘤、哮喘和肺纤维化。总的来说，这些发现突出了所应用方法导致的 EV 特性的变化，并为指导研究人员根据下游功能研究和临床应用选择合适的分离方法提供了令人信服的建议。© 2024 作者。生物技术杂志由 Wiley‐VCH GmbH 出版。

Extracellular vesicles (EVs) operate as chemical messengers that facilitate intercellular communication. Emerging evidence has demonstrated that lung tissue-derived EVs play pivotal roles in pulmonary physiological processes and have potential as biomarkers and therapeutics for lung diseases. Multiple methods have been proposed for the isolation of lung tissue-derived EVs. However, the effects of different tissue pre-treatments on lung EV isolation and subsequent disease biomarker discovery have not yet been comprehensively investigated. In this study, we compared the physical characteristics, recovery yields, and protein compositions of EVs isolated from lung tissues using three methods based on different tissue dissociation principles. Methodologically, the beneficial roles of blood perfusion and gentle meshing were emphasized based on their impact on EV yield and purity. These results demonstrate that different methods enrich distinct subpopulations of EVs that exhibit significant differences in their protein cargo and surface properties. These disparities directly affect the diagnostic detection of marker proteins related to lung diseases, including lung tumors, asthma, and pulmonary fibrosis. Collectively, these findings highlight the variations in EV characteristics resulting from the applied approaches and offer compelling suggestions for guiding researchers in selecting a suitable isolation method based on downstream functional studies and clinical applications.© 2024 The Author(s). Biotechnology Journal published by Wiley‐VCH GmbH.