A Protocol for Exosome Isolation and Characterization: Evaluation of Ultracentrifugation, Density-Gradient Separation, and Immunoaffinity Capture Methods
A Protocol for Exosome Isolation and Characterization: Evaluation of Ultracentrifugation, Density-Gradient Separation, and Immunoaffinity Capture Methods
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DOI：
10.1007/978-1-4939-2550-6_15
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发表时间：
2015-01-01
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影响因子：
--
通讯作者：
Simpson, Richard J.
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文献类型：
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作者：
Greening, David W.;Xu, Rong;Simpson, Richard J.
Exosome是从多种细胞类型释放出来的40-150 nm的胞外小泡，具有多种细胞功能，包括细胞间通讯、抗原递呈以及肿瘤形成蛋白、mRNA和miRNA的转移。外切体是细胞生态位的重要调节者，它们在许多疾病(如癌症)中改变的特征表明它们在诊断和治疗应用以及作为药物输送工具方面的重要性。使用各种策略和技术从生物液和体外细胞培养中纯化外切体。在这一章中，我们揭示了不同于脱落的微泡和凋亡性小泡的外切体的分离、纯化和特征的程序和关键见解。以结直肠癌细胞株LIM1863为细胞模型，对超速离心法(UC-ExOS)、OptiPrep(TM)密度分离法(DG-ExOS)和免疫亲和捕捉法(IAC-ExOS)等外体分离方法进行了综合评价。所有的外切体分离方法均含有40-150 nm的囊泡，免疫印迹法检测外切体标志物(Alix、TSG101、HSP70)呈阳性。该方法使用蛋白质组学方法来表征外切体的蛋白质组成，并使用无标记光谱计数来评估每种方法在外切体分离中的有效性。根据识别的外切体标记和与其生物发生、运输和释放相关的蛋白质的MS/MS谱数，IAC-Exos被证明是分离外切体的最有效方法。然而，当免疫亲和捕获的使用受到限制(由于抗体的可用性和外切体标记的适用性)时，基于密度的分离(DG-Exos)的使用为外切体分离提供了显著的优势。
Exosomes are 40-150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep (TM) density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. All exosome isolation methodologies contained 40-150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers).