Single Extracellular Vesicle Sorting and Analysis

单个细胞外囊泡分选和分析

• 批准号: 10376602
• 负责人: Daniel T Chiu
• 金额: $ 101.78万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376602
• 关键词: Single; Extracellular; Vesicle; Sorting; Analysis

Project Summary Extracellular vesicles (EVs) are membrane-enclosed particles, which are secreted from various cell types into the extracellular space. EVs are highly heterogeneous and comprise a diverse set of surface protein markers as well as intra-vesicular cargoes, such as RNAs. Current approaches to the isolation and study of EVs lack the necessary sensitivity and precision to fully characterize and understand the make-up and the distribution of various EV subpopulations that may be present. In fact, most current EV isolation methodologies, including differential centrifugation, affinity/immuno-magnetic isolation, polymer-based precipitation, size-based exclusion, can be prone to contaminations; for example, affinity/immuno-magnetic methods can be adversely affected by non-specific interactions that can cause co-precipitation of contaminants with EVs. Additionally, these bulk techniques can only report information that is averaged over many millions of EVs, and thus cannot be used to understand the high degree of heterogeneity that exists among the EVs. Yet, understanding the diversity of EVs and the cargos they carry is an essential step towards gaining a better understanding of the precise roles EVs play in both physiological and pathophysiological processes. This understanding, in turn, is important towards realizing the potentials of EVs in diagnostics (e.g. as a new class of biomarkers) and therapeutics (e.g. as drug carriers). To address the limitations of the current methodologies, we propose to develop and apply three sets of new but related technologies for studying EVs with high-throughput at the single-EV level. These new techniques include a nanoscale flow analyzer and flow sorter for the analysis and sorting of individual EVs with high sensitivity and high throughput. Furthermore, we also plan to demonstrate an imaging digital PCR platform for quantifying the RNA contents of single EVs as well as small groups of EVs.

项目概要 细胞外囊泡（EV）是由膜包裹的颗粒，由各种细胞分泌 类型进入细胞外空间。电动汽车具有高度异质性，并包含多种表面 蛋白质标记以及囊泡内货物，例如 RNA。目前的隔离方法 电动汽车的研究缺乏必要的灵敏度和精度来充分表征和理解 可能存在的各种电动汽车亚群的构成和分布。事实上，目前大多数 EV 隔离方法，包括差速离心、亲和/免疫磁隔离、 基于聚合物的沉淀，基于尺寸的排除，可能容易受到污染；例如， 亲和/免疫磁性方法可能会受到非特异性相互作用的不利影响，这些相互作用可以 导致污染物与电动汽车共沉淀。 此外，这些批量技术只能报告数百万个数据的平均信息。 EV，因此不能用来理解不同群体之间存在的高度异质性 电动汽车。然而，了解电动汽车及其所运载货物的多样性是实现这一目标的重要一步 更好地了解电动汽车在生理和心理方面所发挥的精确作用 病理生理过程。反过来，这种理解对于实现潜力非常重要 EV 在诊断（例如作为一类新的生物标志物）和治疗（例如作为药物载体）中的应用。 为了解决当前方法的局限性，我们建议开发和应用三套 用于研究单电动汽车水平高通量电动汽车的新的相关技术。这些新 技术包括纳米级流动分析仪和流动分选机，用于分析和分选个体 具有高灵敏度和高吞吐量的 EV。此外，我们还计划展示成像 用于量化单个 EV 以及小群 EV 的 RNA 含量的数字 PCR 平台。