Foetal bovine serum influence on in vitro extracellular vesicle analyses.

Keywords: cell culture media; EV-depleted fbs; exosomes; extracellular vesicles; foetal bovine serum; in vitro; serum-free media EN cell culture media EV-depleted fbs exosomes extracellular vesicles foetal bovine serum in vitro serum-free media 1 16 16 01/29/21 20210101 NES 210101 INTRODUCTION Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles most notably from either endosomal (i.e., exosomes) or plasma membrane origins (i.e., microvesicles/ectosomes) and released from nearly all mammalian cells (Colombo et al., 2014). Instead, FBS EV-depletion efficiency should be determined by quantifying EV-specific protein markers (e.g., CD9, CD63, CD81) via Western Blot (or proteomic assays) in parallel with unconditioned medium controls, including non-depleted FBS, EV-depleted supernatant, and FBS-EV pellet samples. We believe current best practices should include: 1) using sequential EV isolation protocols based on size and density (i.e., UC/UF/size-exclusion chromatography); 2) extensive characterization of the final EV pellet in terms of size, morphology, RNA, and protein markers to ensure purity of EVs (i.e., tetraspanins) and removal of non-EV-contaminants (i.e., ApoA, ApoB, ApoE); and, 3) inclusion of unconditioned media controls as background reference standards. Importantly, current FBS EV-depletion protocols lack the ability to significantly reduce the quantities of FBS-derived EVs, exRNA species, protein-RNA complex aggregates, and lipoproteins within EV-depleted FBS media, which may contaminate downstream cell-derived EV isolation. [Extracted from the article]