Exosomes represent a subset of small particles secreted by many types of cells under both normal and pathological conditions. The release of microvesicles has demonstrated biological relevance; these particles act as mediators of intercellular communication both within the local microenvironment of the release site as well as systemically. Moreover, since exosomes contain RNA (messenger and miRNA) and protein (membrane-bound and cytosolic) from their cells of origin, and given that this content can be influenced by cell state, they also potentially represent a burgeoning target for biomarker discovery. Critical to understanding the physiological significance of these particles is the development of preparative techniques permitting reliable isolation of purified fractions. While numerous methods of exosome purification exist, including ultracentrifugation, immunoaffinity-based isolation using magnetic beads, precipitation, and ultrafiltration, most are plagued by sample limitations or require long and tedious workflows to achieve success. Here we present a rapid alternative method for the selective fractionation of exosomes from biological samples using an ultrafiltration device. Since the method is spin-based and dependent on size exclusion, the device has broad applications with regards to sample volume and/or type. Optimization of the protocol was aided through use of a mid infrared (MIR)-based spectroscopy platform that permits simultaneous monitoring of lysis conditions, protein quantitation, and analysis of total lipid content during exosome fractionation. Given the ultrafiltration device's capacity for buffer exchange and sample concentration, purified fractions can be easily formatted to meet the requirements of any downstream analysis platform. To demonstrate this, resulting fractions were assayed by numerous techniques including flow cytometry, western blotting, ELISA-based assays, and electron microscopy.
Citation Format: Amedeo Cappione, Sara Gutierrez, Masaharu Mabuchi, Janet Smith, Ivona Strug, Timothy Nadler. A centrifugal ultrafiltration-based method for rapid purification of exosomes from biological samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3483. doi:10.1158/1538-7445.AM2014-3483