Ultracentrifugation is considered the “gold standard” for harvesting microvesicles, though inconsistencies have been reported in reproducibility and repeatability of the data. A standardized method for isolation and assessment of MVs from various body fluids and culture media has not yet been established and hinders reproducible studies for downstream analysis of isolated MVs ( Yuana et al., 2011). MVs are found in different biofluids such as plasma ( Ashcroft et al., 2012), serum ( Dalton, 1975), cultured media (CM Bastida et al., 1984), saliva ( Keller et al., 2011), breast milk ( Hata et al., 2010), amniotic fluid ( Keller et al., 2011), and urine ( Wiggins et al., 1987).Ī variety of methods have been utilized to isolate microvesicles including sucrose gradient, ultracentrifugation, Exoquick™, microfiltration, and immune affinity capture method ( Taylor et al., 2011 Tauro et al., 2012). MVs are also known to carry antigens from microorganisms like viruses and bacteria and can be potential biomarkers for a variety of diseases ( Jayachandran et al., 2011 Raymond et al., 2011). Microvesicles have been extensively studied in serum and culture media from a variety of tumors ( Balaj et al., 2011 Koumangoye et al., 2011) a great body of evidence shows that they can be secreted into the extracellular space and are involved in intercellular communication by transferring functional proteins and RNA molecules between cells ( Skog et al., 2008 Grange et al., 2011 Yang et al., 2011). In this article, we will refer to all types of shed vesicles under the common term of microvesicles (MVs). Their sizes differ from 30 nm in diameter and have been reported up to 5 μm, the former including the more homogenous population of exosomes released from multivesicular bodies (MVBs) and the latter shedding from the plasma membrane which are commonly referred to as MVs ( Di Vizio et al., 2009 Théry et al., 2009). Nanoparticles formed through membrane budding are also called microvesicles and their corresponding process of formation is called microvesiculation ( Muralidharan-Chari et al., 2010). We demonstrate that MVs recovery inversely correlates with viscosity and as a result, sample dilutions should be considered prior to ultracentrifugation when processing any biofluids.Įxosomes are nano-sized vesicles (MVs 30–100 nm) of endosomal origin produced by different parental cells ( Keller et al., 2006 Skog et al., 2008 Muralidharan-Chari et al., 2010). We used different biofluids and spiked them with polystyrene beads and assessed their recovery using the Nanoparticle Tracking Analysis. In this manuscript we addressed the issue of whether viscosity has an effect on sedimentation efficiency of microvesicles using ultracentrifugation. The different chemical and molecular compositions of biofluids have an effect on its viscosity and this could affect movements of the particles inside the fluid. Viscosity is the resistance of a fluid to a deforming force by either shear or tensile stress. Currently, the “gold standard” for isolating microvesicles is ultracentrifugation, although alternative techniques such as affinity purification have been explored. Microvesicles represent the status of the donor cell they are released from and they are currently under intense investigation as a potential source for disease biomarkers. Once released they end up in the systemic circulation and have been found and characterized in all biofluids such as plasma, serum, cerebrospinal fluid, breast milk, ascites, and urine. They are released physiologically under normal conditions but their rate of release is higher under pathological conditions such as tumors. Microvesicles are nano-sized lipid vesicles released by all cells in vivo and in vitro. 6 Department of Developmental Biology, Harvard School of Dental Medicine, Boston, MA, USA.5 Exosome Diagnostics Inc, New York, NY, USA.4 Biopolymers Facility, Harvard Medical School, Boston, MA, USA.
3 Neuro-Oncology Research Group, Cancer Center Amsterdam, Amsterdam, Netherlands.2 Department of Neurology and Radiology, Massachusetts General Hospital, Boston, MA, USA.1 Harvard Catalyst Laboratory for Innovative Translational Technologies, Harvard Medical School, Boston, MA, USA.Johan Skog 5 and Winston Patrick Kuo 1,6*
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