Comparative small RNA reads from human serum extracellular vesicles isolated from healthy volunteers and sepsis patients by different methods

Extracellular vesicles (EVs) are intercellular communicators with key functions in physiological and pathological processes, and have recently garnered interest because of their diagnostic and therapeutic potential. The past decade has brought about the development and commercialization of a wide array of methods to isolate extracellular vesicles from serum. Which subpopulations of EVs are captured strongly depends on the isolation method, which in turn determines how suitable resulting samples are for various downstream applications. To help clinicians and scientists choose the most appropriate approach for their experiments, isolation methods need to be comparatively characterized. Few attempts have been made to comprehensively analyze vesicular microRNAs (miRNAs) in patient biofluids for biomarker studies. To address this discrepancy, we set out to benchmark the performance of several isolation principles for serum EVs in healthy individuals and critically ill patients. Here, we compared five different methods of EV isolation regarding their suitability for biomarker discovery-focused miRNA sequencing as well as biological characteristics of captured vesicles. Our findings reveal striking method-specific differences in both the properties of isolated vesicles and the ability of associated miRNAs to serve in biomarker research. While isolation by precipitation and membrane affinity was highly suitable for miRNA-based biomarker discovery, methods based on size-exclusion chromatography failed to separate patients from healthy volunteers. Isolated vesicles differed in size, quantity, purity and composition, indicating that each method captured distinctive populations of EVs as well as additional contaminants. Even though the focus of this work was on transcriptomic profiling of EV-miRNAs, our insights also apply to additional areas of research. We provide guidance for navigating the multitude of EV isolation methods available today, and help researchers and clinicians make an informed choice about which strategy to use for experiments involving critically ill patients.

细胞外囊泡（Extracellular vesicles, EVs）是一类关键的细胞间通讯信使，在生理与病理过程中发挥核心功能，近年来因其在诊断与治疗领域的应用潜力受到学界广泛关注。过去十年间，诸多可从血清中分离细胞外囊泡的方法已完成开发并实现商业化。所捕获的细胞外囊泡亚群类型很大程度上取决于所采用的分离方法，而分离方法又直接决定了所得样本在各类下游应用中的适配性。为帮助临床医生与科研人员为实验选择最适宜的分离策略，需对各类分离方法开展系统性比较表征。此前鲜有研究针对生物标志物研究场景，全面分析患者生物体液中的囊泡微小RNA（microRNAs, miRNAs）。为填补这一研究空白，我们旨在对健康个体与重症患者血清中细胞外囊泡的数种分离原理的性能进行基准测试。本研究针对五种不同的细胞外囊泡分离方法，就其适配以生物标志物发现为导向的miRNA测序的能力，以及所捕获囊泡的生物学特性展开了比较分析。研究结果显示，不同分离方法在分离所得囊泡的物理属性，以及相关miRNA用于生物标志物研究的效能上均存在显著差异。其中，基于沉淀法（precipitation）与膜亲和法（membrane affinity）的分离方式可高效适配基于miRNA的生物标志物发现工作，而基于尺寸排阻色谱法（size-exclusion chromatography）的分离方法则无法区分重症患者与健康志愿者群体。分离得到的囊泡在尺寸、数量、纯度与组成上均存在显著差异，表明每种方法所捕获的细胞外囊泡亚群及伴随的污染物均具有特异性。尽管本研究的核心聚焦于细胞外囊泡miRNA的转录组谱分析，其研究结论同样适用于其他相关研究领域。本研究为当前市场上众多的细胞外囊泡分离方法提供了科学选择指南，助力科研人员与临床医生为涉及重症患者的实验做出明智的策略选择。