Date Published: January 23, 2017
Publisher: Public Library of Science
Author(s): Inas Helwa, Jingwen Cai, Michelle D. Drewry, Arthur Zimmerman, Michael B. Dinkins, Mariam Lotfy Khaled, Mutsa Seremwe, W. Michael Dismuke, Erhard Bieberich, W. Daniel Stamer, Mark W. Hamrick, Yutao Liu, Giovanni Camussi.
Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 μl and 50 μl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40–150 nm). The three kits, though, produced a significantly higher yield (80–300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available.
Extracellular vesicles are spherical particles with phospholipid bilayers released by various cell types in vivo into body fluids such as serum, urine, cerebrospinal fluid, breast milk, aqueous humor, and amniotic fluid [1–7], as well as in vitro by cultured cells . It is becoming increasingly obvious that these vesicles are pivotal mediators of cell-cell communication in multicellular organisms, having pleiotropic cellular and biological functions [9–14]. Hence, they are now regarded as multifunctional signaling complexes and major contributors to disease pathways such as tumor progression and metastasis . Generally, extracellular vesicles are classified according to their cellular origin and biogenesis into microvesicles, exosomes, and apoptotic bodies . Exosomes range in size from 40–150 nm, and they are derived from the endosomal compartment within the cell . Exosomal content includes genomic DNA, RNA, proteins, and lipids [10, 13, 15, 18, 19]. Over the past decade, exosomes have gained specific interest as microRNA (miRNA) carriers, disease biomarkers, and potential therapeutic targets [17, 20, 21]. Despite their importance, exosome isolation and characterization are still considered major scientific challenges [22, 23], and identifying the optimal technique to isolate exosomes is essential for further biomarker discoveries.
In this study, we compared four different exosome isolation techniques (UC, ExoQuick, miRCURY, and TEIR) using six different starting volumes (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) of a pooled human serum, as well as three different volumes (1 ml, 500 μl and 100 μl) of six individual human donor samples. Using TEM imaging and NTA, we confirmed that all four technologies isolated particles within the size range of exosomes (40–150 nm) and with the traditionally reported morphology [43, 57, 58]. We further confirmed the identity of these particles as exosomes by analyzing the expression of exosome-enriched proteins, CD9 and CD63, through TEM immunogold staining and western blot. Our data showed that all samples expressed CD63 and CD9 similar to previous publications . Quantitatively, we compared the exosomes isolated using the different techniques in terms of the physical properties of the particles, the particle yield, and the quality and quantity of their exRNA content. Overall, our study supports the feasibility of using these four methods to isolate large number of exosomes from different volumes of human serum samples. To our knowledge, this is the first study to integrate zeta potential and absolute miRNA quantification, using ddPCR as isolation technique comparison parameters.