Enabling isolation and characterization of single extracellular vesicles and their molecular contents using multi-marker surface signatures

摘要 细胞外囊泡（EVs）被认为是转运RNA的重要途径 和其他细胞间的信号分子。人体内大多数类型的细胞分泌 每个EV都可能具有不同的生物功能。研究不同类型的电动汽车 以及它们在正常生理功能和疾病相关过程中的作用，需要可靠的 从血液或尿液等容易获得的体液中捕获这些颗粒的方法。我们 我们的目标是开发一种新的方法来分离特定类型的EV， 他们的表面。我们的假设是我们可以利用两个或多个特定分子的存在 在由特定细胞类型分泌的选择EV的表面上。一旦我们分离出高纯度的 我们可以更容易地识别和测量它们的分子含量。 我们将开发一种新的可扩展的方法来识别表面的组合 存在于来自特定细胞类型的EV上的分子，并使用其来鉴定多标记物 已知几种类型的细胞的“表面特征”将EV直接分泌到细胞中。 血流我们将开发一种新的方法来隔离这些表面的EV 签名.这种方法，这将只捕捉电动汽车具有所有的目标表面， 标记物，应该大大提高捕获特异性，因此，解决了一个主要的 现有EV隔离方法的缺点。 我们将通过测量每一种药物的存在来验证分离的EV的纯度。 使用最近开发的多标记物测定和 新型高灵敏度单EV表征仪器。生产高纯度的电动汽车， 特定的细胞类型将使EV的研究比目前更有针对性 执行。例如，我们将在纯化的EV中鉴定特定的调节RNA分子， 并测量这些分子在单个EV上的分布 水平使用新的仪器和分析技术。最后，我们将改进隔离 能够从几种常用的生物流体中高通量分离EV的方法， 自动化单个EV表征仪器，以实现目前 要么是不可能的，要么是太耗时而不符合成本效益。

Abstract Extracellular vesicles (EVs) are believed to be an important means of transporting RNA and other signaling molecules between cells. Most types of cells in the human body secrete EVs which are each likely to have distinct biological functions. Studying different types of EVs and their role in normal physiologic function and disease-related processes requires a reliable means of capturing these particles from readily accessible body fluids, like blood or urine. Our goal is to develop a new way to isolate specific types of EVs based on the molecules present on their surface. Our hypothesis is that we can use the presence of two or more specific molecules on the surface of select EVs secreted by a particular cell type. Once we isolate highly purified populations we can more easily identify and measure their molecular contents. We will develop a new scalable approach to identifying combinations of surface molecules present on EVs from particular cell types and use this to identify multi-marker “surface signatures” for several types of cells known to secrete EVs directly into the bloodstream. We will develop a new method to isolate the EVs with each of these surface signatures. This approach, which will only capture the EVs having all of the targeted surface markers, should greatly improve the capture specificity, thus, addressing one of the main shortcomings of the existing EV isolation methods. We will verify the purity of the isolated EVs by measuring the presence of each of the surface-signature markers on every EV using recently developed multi-marker assays and a new high-sensitivity single-EV characterization instrument. Generating highly purified EVs from specific cell types will enable more targeted studies of EVs than those that are presently performed. For example, we will identify specific regulatory RNA molecules in the purified EVs by next generation sequencing and measure the distribution of these molecules at the single EV level using the new instrumentation and assay techniques. Lastly we will refine the isolation methods to enable high-throughput isolation of EVs from several commonly used biofluids and automate the single EV characterization instrumentation to enable large studies that presently would be either impossible or too time-consuming to be cost-effective.