Isolation and characterization of exosomes from cell culture supernatants and biological fluids
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.
- SourceAvailable from: Yuxuan Miao
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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. "
ABSTRACT: Vertebrate cells have evolved elaborate cell-autonomous defense programs to monitor subcellular compartments for infection and to evoke counter-responses. These programs are activated by pathogen-associated pattern molecules and by various strategies intracellular pathogens employ to alter cellular microenvironments. Here, we show that, when uropathogenic E. coli (UPEC) infect bladder epithelial cells (BECs), they are targeted by autophagy but avoid degradation because of their capacity to neutralize lysosomal pH. This change is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized to lysosomes. TRPML3 activation then spontaneously initiates lysosome exocytosis, resulting in expulsion of exosome-encased bacteria. These studies reveal a cellular default system for lysosome homeostasis that has been co-opted by the autonomous defense program to clear recalcitrant pathogens. Copyright © 2015 Elsevier Inc. All rights reserved.Cell 05/2015; 161(6). DOI:10.1016/j.cell.2015.05.009 · 33.12 Impact Factor
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- "It was recently found that biological fluids, including malignant effusions, contain a high number of exosomes : small membrane vesicles of endocytic origin with a size range of 30 to 150 nm  . These contain various kinds of proteins promoting cancer progression and metastatic spread, including growth factors, cytokines and chaperones    . These biologically active substancies can mediate cross-talk between malignant cells and other cells which belong to the tumor microenvironment. "
ABSTRACT: The majority of tumor-related deaths are due to metastasis. Despite the clinical importance of understanding metastasis, we lack knowledge of the molecular mechanisms underlying tumor cell spreading and cell survival far from the primary tumor. Elucidating the molecular characteristics of highly metastatic carcinoma cells would help identify biomarkers or therapeutic targets relevant to predicting or combatting metastasis, and for this the phenotype of metastatic cells could be much more important than their genotype. Hence, proteomic approaches have wide potential utility. This review discusses possibilities of analyzing metastasis-specific protein patterns in a range of sample types, including in vitro and in vivo cancer models, and tissues and biological fluids from patients. Proteome approaches can identify proteins involved in regulating the metastatic capacities of tumors. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.PROTEOMICS - CLINICAL APPLICATIONS 04/2015; DOI:10.1002/prca.201400128 · 2.68 Impact Factor
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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. "
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.Acta histochemica et cytochemica official journal of the Japan Society of Histochemistry and Cytochemistry 12/2014; 63(3). DOI:10.1369/0022155414564219 · 1.22 Impact Factor
Questions & Answers about this publication
- 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.
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.Following
- 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?
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!Following