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Isolation and characterization of exosomes from cell culture supernatants and biological fluids

Institut Curie, Paris, France.
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 05/2006; Chapter 3(Unit 3):Unit 3.22. DOI: 10.1002/0471143030.cb0322s30
Source: PubMed

ABSTRACT Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.

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    • "Exosomes are typically derived from the inward budding of multivesicular bodies (MVBs) and are of interest because they serve as highly efficient export vehicles (Thé ry et al., 2002). Immunofluorescent staining of EUCVs revealed colocalization of several proteins frequently found in exosomes, including the ESCRT proteins Alix and Tsg101, as well as the tetraspanin CD63 (Thery et al., 2006) (Figure 2A). To more quantitatively confirm the presence of exosome markers on EUCVs, the bacteria were first covalently linked to magnetic beads before exposure to BECs (Lö nnbro et al., 2008), and EUCV-encased bacteria were isolated from cell-free medium on a magnetic rack. "
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    • "It was recently found that biological fluids, including malignant effusions, contain a high number of exosomes [30]: small membrane vesicles of endocytic origin with a size range of 30 to 150 nm [30] [31]. These contain various kinds of proteins promoting cancer progression and metastatic spread, including growth factors, cytokines and chaperones [31] [32] [33] [34]. These biologically active substancies can mediate cross-talk between malignant cells and other cells which belong to the tumor microenvironment. "
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    • "Due to its easy accessibility, saliva has become a potential source for exosomal biomarkers for diagnostic and prognostic assessments (Lau et al. 2013). The most accepted method for isolation of exosomes in general and of salivary exosomes in particular is based on ultracentrifugation (UC) (Théry et al. 2006). Although this technique is believed to obtain minimally contaminated pellets of exosomes, it demands a very complicated and prolonged process that utilizes specialized equipment. "
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    ABSTRACT: ExoQuick-TCTM (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC.
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Questions & Answers about this publication

  • Jan Van Deun added an answer in Exosomes:
    Any suggestions on the approaches to study exosome?

    Specifically how can I isolate exosome from condition medium and ascites, how to break down exosome and study the composition of membrane (such as lipid, protein) as well as contents (miRNA, DNA). Thank you very much.

    Jan Van Deun · Ghent University

    Hi Yilin,

    The "classic" paper on exosome isolation and characterization is the one from Théry et al. (Isolation and characterization of exosomes from cell culture supernatants and biological fluids), of which I've added the link below to request a full text.

    I have recently published another study on isolation methods and the resulting purity myself, where you can also find some protocols for protein and RNA analysis. This paper is open access (second link). Based on these results I would advise to stay away from commercial kits if you can. For the highest purity, go for an OptiPrep or sucrose density gradient.

  • Kenneth W Witwer added an answer in Exosomes:
    Did anyone isolate exosomes for microRNA extraction from MSC or any other cell lines?

    If you did,what is the best method to isolate exosomes for microRNA extraction?  How much culture media  need to collect or how many cells need to culture?

    Thanks!

    Kenneth W Witwer · Johns Hopkins Medicine

    Remember to use completely vesicle-free media, preferably serum-free, as you will otherwise isolate bovine RNA that for the most part cannot be distinguished from RNA of your species of interest. If your goal is to isolate exosomes, you will not obtain reasonably pure exosomes by doing only a slow first spin to remove cells. At a minimum, sequential centrifugations at higher g are needed to remove cell debris, apoptotic bodies, and larger vesicles. Showing that a putative exosomal marker is present is also not the same thing as demonstrating that co-purifying complexes are absent!