用 Superose 6 尺寸排阻色谱法分离人血中的小细胞外囊泡 (sEV)。

IF 15.5 1区 医学 Q1 CELL BIOLOGY
Jerome Nouvel, Gonzalo Bustos Quevedo, Tony Prinz, Ramsha Masood, George Daaboul, Tanja Gainey-Schleicher, Uwe Wittel, Sophia Chikhladze, Bence Melykuti, Martin Helmstaedter, Karl Winkler, Irina Nazarenko, Gerhard Pütz
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引用次数: 0

摘要

细胞外囊泡(EV)是液体活检的重要目标。然而,将基于 EV 的生物标记物引入临床实践的尝试并未取得预期的成功。失败的原因之一是缺乏从复杂生物流体(如无细胞血浆或血清)中进行 EV 基线纯化的可靠方法。由于现有的一步式EV分离方法不足以纯化EV,大多数临床样本研究要么是针对EV和脂蛋白的混合物进行的,而EV的真实数量和它们各自的特定生物标记物含量仍然难以确定;要么是针对数量较少、体积足够大的样本进行的,从而无法按大小和密度进行精细的两步式EV分离,结果是纯度很高,但回收率极低。在这里,我们介绍了使用 Superose 6 作为基质的快速蛋白质液相色谱法(FPLC),以从几乎不含可溶性蛋白质和脂蛋白的生物流体中获得小型 EV。在估算人体样本中小EV的实际数量的同时,我们还展示了餐后阶段持续时间对纯化EV中脂蛋白比例影响的时间分辨率,为建议在人体研究中使用空腹样本提供了可接受的时间框架。此外,我们还评估了纯EVs在液体活检中的潜在价值,例如检测了从胰腺癌患者血清中分离出来的纯FPLC衍生馏分中的EV和肿瘤生物标记物。不同技术的研究结果表明,纯EVs中存在与疾病相关的生物标记物,这支持了将单颗粒分析用于液体活检的可行性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography

Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography

Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV-based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell-free plasma or serum. Because available one-step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2-step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV- and tumour-biomarkers in pure FPLC-derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases-associated biomarkers in pure EVs, supporting the feasibility of using single-vesicle analysis for liquid biopsy.

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来源期刊
Journal of Extracellular Vesicles
Journal of Extracellular Vesicles Biochemistry, Genetics and Molecular Biology-Cell Biology
CiteScore
27.30
自引率
4.40%
发文量
115
审稿时长
12 weeks
期刊介绍: The Journal of Extracellular Vesicles is an open access research publication that focuses on extracellular vesicles, including microvesicles, exosomes, ectosomes, and apoptotic bodies. It serves as the official journal of the International Society for Extracellular Vesicles and aims to facilitate the exchange of data, ideas, and information pertaining to the chemistry, biology, and applications of extracellular vesicles. The journal covers various aspects such as the cellular and molecular mechanisms of extracellular vesicles biogenesis, technological advancements in their isolation, quantification, and characterization, the role and function of extracellular vesicles in biology, stem cell-derived extracellular vesicles and their biology, as well as the application of extracellular vesicles for pharmacological, immunological, or genetic therapies. The Journal of Extracellular Vesicles is widely recognized and indexed by numerous services, including Biological Abstracts, BIOSIS Previews, Chemical Abstracts Service (CAS), Current Contents/Life Sciences, Directory of Open Access Journals (DOAJ), Journal Citation Reports/Science Edition, Google Scholar, ProQuest Natural Science Collection, ProQuest SciTech Collection, SciTech Premium Collection, PubMed Central/PubMed, Science Citation Index Expanded, ScienceOpen, and Scopus.
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