Exosomes as a potential tool for a specific delivery of functional molecules.

Extracellular membrane vesicles derived from the endosomal compartments and released by the fusion of the multivesicular bodies with the cell membrane are referred as exosomes (Exo) [Van Niel et al., J Biochem 140:13-21, 2006]. They function as mediators of intercellular communication and are employed by the organism in the regulation of systemic and local processes. Meantime, Exo are recognized as an indispensable entity of physiological fluids [Caby et al., Int Immunol 17:879-887, 2005; Lasser et al., J Transl Med 9:9, 2011; Lasser et al., Am J Rhinol Allergy 25:89-93, 2011]. Exo and other types of extracellular vesicles, e.g., exosome-like vesicles [van Niel et al., Gastroenterology 121:337-349, 2001] and microvesicles (MV) [Daveloose et al., Thromb Res 22:195-201, 1981], contain multiple functional molecules including lipids [Vidal et al., J Cell Physiol 140:455-462, 1989]; proteins [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; mRNA [Valadi et al., Nat Cell Biol 9:654-659, 2007]; DNA [Waldenstrom et al., PLoS One 7:e34653, 2012]; noncoding RNA, e.g., miRNA [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; and retrotransposon elements [Balaj et al., Nat Commun 2:180, 2011]. Assessment of the biological functions of Exo showed that they deliver specifically their cargo from the donor to recipient cells. Albeit the molecular mechanisms of this process are not fully understood, approaches for the application of Exo and MV as a tool for a cell-specific delivery of signalling molecules were successfully tested in in vitro and in vivo models [Maguire et al., Mol Ther 20:960-971, 2012]. Ovarian cancer cells release Exo, which bind stroma cells as well as donor cancer cells [Escrevente et al., BMC Cancer 11:108, 2011]. Here we describe an experimental approach for the assessment of Exo interaction and uptake by target cells. Methods for the isolation and purification of Exo from cell culture supernatants are included. To allow visualization of vesicle uptake, labelling of Exo with different fluorescent dyes, such as CFSE, PKH, DHPE, and DiOC18, is presented. Finally, we explain qualitative and quantitative analysis of Exo uptake by immunofluorescence and flow cytometry, respectively.