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

摘要 细胞外小泡被认为是运输rna的一种重要方式。 和其他细胞间的信号分子。人体内大多数类型的细胞都能分泌 电动汽车，每一种都可能具有不同的生物功能。研究不同类型的电动汽车 而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接触到的体液中捕获这些颗粒的方法。我们的 目标是开发一种新的方法来分离特定类型的电动汽车，基于存在的分子 它们的表面。我们的假设是，我们可以利用两个或更多特定分子的存在 在特定细胞类型分泌的精选电动汽车的表面。一旦我们分离出高度纯化的 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曲面的组合 EV上存在的来自特定细胞类型的分子，并利用这一点来识别多标记 几种已知能将EV直接分泌到细胞内的细胞的“表面特征” 血液流动。我们将开发一种新的方法来分离每个表面的电动汽车 签名。此方法将仅捕获具有所有目标表面的电动汽车 标记物，应大大提高捕获的特异性，从而解决主要问题之一 现有电动汽车隔离方法的不足之处。 我们将通过测量每一辆电动汽车的存在来验证隔离电动汽车的纯度 使用最近开发的多标记分析和一种 新型高灵敏度单电动汽车特性测试仪。生产高纯度的电动汽车 特定的细胞类型将使电动汽车的研究比目前的研究更有针对性 已执行。例如，我们将在纯化的电动汽车中鉴定特定的调节rna分子。 通过下一代测序并测量这些分子在单个电动汽车上的分布 使用新的仪器和化验技术。最后，我们将改进隔离 从几种常用的生物液体中高通量分离肠道病毒的方法和 使单个电动汽车表征仪器自动化，以实现目前 要么是不可能的，要么是太耗时了，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.