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The presence of air bubbles could induce acquisition of artifacts. Ensure that sheath fluid is sterile and that 0. All cleaning solutions should be freshly prepared and filtered with 0. All acquisitions have to be performed using sterile FACS tubes. The sample flow rate has to be as slow as possible. To avoid formation of bubbles, the sheath tank should be filled the day before the experiment, and fluidic startup must be the first operative configuration performed. All parameters are statistically paired robust standard deviation, or rSD with a multiparameter baseline three drops of same bead suspension in 0.

In general, the cleanliness of the instrument should be frequently checked. Select and check fluorescence channels i. It is recommended to select fluorochromes that emit at a greater distance to reduce spillover phenomena in adjacent fluorescence channels. Due to Rayleigh scattering Shapiro, , smaller particles have a size similar to the wavelength of the blue laser nm , which defines physical characteristics.

It is recommended to select fluorescent beads and fluorescent dye used to detect intact EVs detected in the same fluorescence channel e. Select threshold on the fluorescence channel instead of physical scatter. This is strongly recommended. This will allow delineation of the gate s of interest in the desired size range Fig. In order to avoid formation of aggregates, centrifuge the intermediate CFSE solution for 30 sec at 10, rpm.

The tube has to be considered as the correct control to verify CFSE specificity. Load a tube containing a selected EV fraction suspension see step 15 stained with CFSE as in step 17 but at room temperature to set correct dimensional gate Fig. Draw a histogram for each fluorescence channel and verify the absence of spillover, autofluorescence, and nonspecificity. The accurate titration of antibodies and the use of related isotype controls are critical steps for successful implementation of the FCM procedure. The use of compensation particles [e. TruCount and sample preparation must be very accurate, with mixing by repeated pipetting to avoid clump formation and volume loss.

During traditional EV isolation, discarding the pellet after 10K centrifugation or filtering the supernatant with a 0. For this reason, it is becoming increasingly important to develop and optimize useful protocols to isolate the different subpopulations, avoiding the possibility of losing subtypes. Several methods have been adopted to count and characterize EVs e.

All the methodologies present difficulties and limitations, ranging from low sensitivity to very high cost to poor statistical validation. Some critical issues may affect different aspects of the protocols presented in this unit. One of these is the starting amount of vesicles Basic Protocol 1 necessary to perform downstream analysis Basic Protocol 2. Indeed, we have observed that primary cultures release fewer vesicles than cell lines do. As mentioned in Basic Protocol 1 , we perform the experimental procedure with polypropylene tubes instead of the widely used polyallomer tubes.

Moreover, sample preparation for FCM Basic Protocol 2 must be systematic: the choice and purity of buffers, the concentrations of particles, the fluorescent dyes used to discriminate intact vesicles, and the titration of the antibodies as well as the isotype controls have to be considered with great care. In Basic Protocol 2 , it is also very important to check the injection pressure of the sample in the chamber sample flow rate and the pressure of the transport liquid sheath flow pressure , which creates the laminar flow.

Both pressures must be as low as possible in order to slow down the flow of the vesicles. A troubleshooting guide is presented in Table 1. This unit has highlighted the critical and key points in the basic protocols. If the protocols are followed as indicated, optimal results are warranted. These protocols can be adapted to isolate and characterize EV subpopulations from cell types other than MSCs.

Volume 48 , Issue 1. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account. If the address matches an existing account you will receive an email with instructions to retrieve your username. Webinars Videos. Invagination into endosome to form multi-vesicular bodies MVBs. MVBs fuse with plasma membrane, then exosomes are secreted into extracellular space. Microvesicles are released from plasma membrane with surface protein from cells.

Exosomes and microvesicles are affiliated with EVs. EVs derived from cells contribute to cell communication, cell signaling and diagnosis. Characteristics of exosomes and microvesicles do not clearly distinguish them. Some studies have tried to discriminate exosome and microvesicle based on origin, size and density [ 8 ].

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Exosomes and microvesicles can be distinguished based on size, but cannot be easily distinguished based on only protein markers on the vesicle membrane. Tetraspanins, which are commonly used to define EVs, are enriched in both exosomes and microvesicles [ 11 , 12 ]. Distinction between the two classes requires more-accurate clues than tetraspanin presence. However, this classification based on biogenesis can cause confusion, because small microvesicles that arise from evagination of plasma membranes are also called exosomes in some cases, and because different groups of EVs cannot be completely discriminated during isolation [ 5 , 16 ].

Sometimes EVs are named after the cells that released them; for example, vesicles released from cardiomyocytes are called cardiosomes [ 17 ], vesicles from prostate epithelial cells are called prostatosomes [ 18 ], and vesicles released from tumor cells are called oncosomes [ 19 ]. When an EV contacts a cell, their plasma membranes merge; as a result, the contents of the EV enter the cell, and can affect its function [ 21 ].

Proteins that are associated with EVs also have functions in the microenvironment. For example, the tetraspanins CD81 and CD9 immunoprecipitate together. EVs can contribute to tumor progression by facilitating angiogenesis while suppressing immune responses [ 22 , 23 , 24 ]. Functional proteins are released in association with EVs, so they are also involved in many neurological processes [ 25 , 26 ]. EVs are found in most body fluids e. An EV contains information that is related to its cell of origin, so isolating and analyzing EVs from body fluids can give important clues to diagnosis and prognosis of disease [ 29 ].

Profiles of miRNAs within serum EVs from ovarian cancer patients are significantly different than those of normal patients and of patients with benign cancer [ 30 ]. Proteins in urinary EVs may provide biomarkers of acute kidney injury; for example, fetuitin-A in urinary EVs may be useful in diagnosing structural renal injury [ 32 , 33 ], and decrease in the level of podocalyxin-like protein 1 PODXL in urinary EVs may provide a biomarker to classify renal disease [ 34 ].

However, in addition to EVs, body fluids contain diverse molecular components that can impede accurate and efficient analysis [ 35 ]. Furthermore, EVs are not abundant in biological fluids, so their contents are not easy to analyze [ 38 ]. Conventional isolation by ultracentrifugation recovers an average 0. Altogether, the presence of other particles, the scarcity of EVs, and their contents hinder efficient analysis of EVs and complicate subsequent procedures that use them.

Accordingly, to improve the yield and purity of EVs, and to ensure that subsequent analyses are not disturbed, efficient isolation of EVs is important Fig.

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Outline of the workflow for procedure for isolation and analysis of EVs. EVs are isolated from body fluid or cell culture medium CCM. Various methods have been developed to isolate EVs effectively from biological fluids by exploiting a characteristic of EVs to separate them from coexisting particles. The methods differ in yield, purity and size distribution of isolated EVs.

Therefore, high-quality EVs must be isolated using a method that is appropriate to the aim of the study, and is compatible with subsequent analyses [ 8 , 38 ]. The following sections discuss the merits and demerits of various techniques to isolate EVs. EVs sink due to the centrifugal force and form a pellet at the bottom in an ultracentrifuge tube [ 44 ]. Ultracentrifugation: differential ultracentrifugation and density gradient centrifugation. Ultracentrifugation is a centrifugation with high g-force. Ultracentrifugation is commonly used to isolate EVs; density gradient centrifugation isolates EVs with fewer impurities and aggregation.

This is the most-commonly-used protocol, but must be modified when the EVs are suspended in viscous fluids such as plasma or saliva [ 50 ]. The quantity of collected EVs is affected by centrifuging speed and time, so these parameters must be optimized for each rotor type. The density fraction in which EVs accumulate is affected by gradient material [ 46 , 52 ], and by the source of EVs [ 47 ]. For example, EVs from saliva accumulate in fractions with higher densities than EVs from conditioned medium [ 47 ].

Results can provide a diagnostic standard for comparison of patients with healthy individuals [ 54 ]. CD24 is present in serum EVs of breast-cancer patients, whereas EpCAM is not, so this difference may be a way to discriminate breast cancer from others [ 54 ]. Another disadvantage is that protein contaminants co-occur with EVs isolated by this method, so use of a Bradford assay to estimate EV amount is not reliable [ 44 , 51 , 57 ].

Moreover, EVs isolated at high rotation speed have different phenotype, size, and even surface proteins than those isolated at low rotation speed. Hence, EVs isolated by density gradient centrifugation have good purity and intact morphological characteristics [ 56 , 61 ]. However, isolation based on size cannot discriminate EVs from other small vesicles, or among subpopulations of similarly-sized EVs. Immunoaffinity exploits interactions between antibodies and surface proteins of EVs to isolate EVs. Antibodies specific to surface proteins of EVs e.

EVs isolated based on immunoaffinity have different characteristics than those isolated based on size. Size-based separation cannot distinguish among subpopulations of EVs. Many studies have discovered EV biogenesis and subpopulations by exploiting the interaction of surface proteins with specific antibodies. Tetraspanin-specific antibodies are common in immunoaffinity [ 65 ]; for example, CD81 is internalized more slowly than CD9 [ 66 ].

The EGFR exosomal proteins are possible diagnostic biomarkers in immunoaffinity methods [ 65 ]. Immunoaffinity has been incorporated into hybrid approaches that use more than one isolation method [ 68 , 69 , 69 ]. EVs can be isolated using magnetic beads coated with antibodies in a minimal volume of plasma [ 54 ]. Isolation of the magnetic beads enriches EVs Fig. Surface plasmon resonance imaging SPRi with antibody microarrays detects specific proteins on EV membranes in tumor-cell culture medium [ 69 ].

EVs isolated by the device have positive correlation with tumor cell in metastasis and prognosis prediction.

Suresh Mathivanan - Isolation and characterization of exosomes and ectosomes

Isolation method using antibody-conjugated bead. Interaction with antibody and surface protein in EV is a key factor in immunoaffinity. Immunoaffinity method enables identification of proteins including positive markers in EVs. When immunoaffinity is used to isolate EVs, subpopulations of EVs can be identified by sorting them according to their specific surface proteins. However, antibodies are expensive, so this method is not appropriate for large samples. Moreover, for subsequent experiments, EVs must be displaced from the beads; this step may damage the EVs. Recent studies have classified EV by size rather than by surface protein.

Use of immunoaffinity does not identify the source of EV exosome, ectosome, apoptotic body , so this method is not suitable for sorting by size. For example, exosomes, microvesicles and apoptotic bodies all have EpCAM on the membrane [ 8 ]; immunoaffinity detects the same exosomal marker in these vesicles, but they have different characteristics. Future studies should develop immunoaffinity methods to correlate surface protein identity with EV functions and properties. Size differences can be exploited to isolate EVs.

In general, two types of size exclusion are used: filtration and chromatography Fig. Filtration captures EVs on membranes, while allowing small particles like proteins to pass through them [ 72 ]. This method requires appropriate choice of pore size. Size-exclusion with a filter and b chromatography. Sorting targeted-sized EVs by size exclusion. Filter-based isolation requires sorting proteins and EVs.

The device with electrophoretic migration processes and size exclusion isolates EVs. Small particles enter the pore and move slowly, whereas large particles pass through the column rapidly. EV-containing solution is collected. Pore size of 0. The EV level in smokers is decreased by early apoptosis. EVs can be isolated by combining electrophoretic migration processes and size exclusion nm pore size [ 73 ]; a nanoporous membrane allows small impurities to pass through into the flow, but retains EVs larger than membrane pore size.

Size-exclusion chromatography SEC uses a column packed with beads that have pores smaller than EVs of concern [ 74 ]. Particles smaller than pore size enter them and move slowly, whereas particles larger than pore size pass around beads and exit the column rapidly.

Fractions containing samples are eluted sequentially in order of decreasing size; the EV-containing fraction can be collected and analyzed. However, filter-based isolation and SEC separate components by their size, and therefore cannot discriminate particles that have similar size but different characteristics. One important requirement of EV isolation is to remove high-density lipoproteins HDL as well as proteins. EVs are highly isolated in fractions 9— HDL in fractions 9—12 is just 0. EVs isolated by chromatography are used in diagnosis [ 78 , 79 , 79 ]. Ovarian adenoma can be diagnosed in serum EVs isolated by chromatographic isolation; serum EVs are analyzed using NTA and western blotting.

SEC easily isolates EVs from concentrated body fluid in one step, and has applications in diagnosis of some diseases [ 78 ]. Various methods that use polymers have been developed to ease the isolation process and reduce the isolation time [ 80 ]. The methods use polymers that can precipitate or displace EVs according to surface characteristics.

These proteins are demonstrated in presence of ACS. EVs move toward the dextran phase, which has surface characteristics that are favorable to EVs [ 83 ].

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ATPS is quick and easy, and does not requiring any incubation process [ 84 ]. However, as EVs are isolated in a dextran-containing solution, so the effect of dextran must be reduced before ATPS can be widely accepted. Scheme of aqueous two phase system ATPS separation. EVs move into a dextran phase that has surface properties that are favorable to EVs. ATPS purifies EVs based on precipitating properties from sample without expensive equipment or large samples.

Protein organic solvent precipitation PROSPR is an EV precipitation method that is a fast and simple process with organic solvents acetone, chloroform, trichloroacetic acid [ 86 ]. Biological fluids contain numerous proteins, soluble factors, and lipoproteins [ 37 ]. Removal of protein from biological fluids is an important requirement in EV analysis. Proteins that have hydrophilic and hydrophobic regions have dielectric strength in aqueous solution. The organic solvent is attracted to oppositely-charged amino acid residues and promote protein aggregation. Moreover, organic solvent with a salt improves protein removal [ 87 ].

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EVs can be isolated by microfluidic devices that induce flow of liquids within micro-sized channels [ 38 , 89 ]. Microfluidic devices are small but purify and separate faster than other isolation methods. Microfluidic devices require only a small sample, so the cost, processing time, and consumption of reagents are reduced. EVs isolated using microfluidic devices tend to retain their morphology. Microfluidics techniques capture EVs by using immunoaffinity, by sieving, or by trapping them in porous structures [ 90 ]. Microfluidic devices have been combined with immunoaffinity methods to detect diagnostic markers.

EVs are captured by binding to specific antibodies on channels or by passing through membranes Fig. The antibodies bind specific EVs because EVs secreted by tumor cells have surface specific markers. Antibodies coated on the surface of device increase the efficiency of EV capture. Microfluidic system to isolate EVs.

Microfluidics techniques developed for EV purification are classified into immunoaffinity approach, sieving EVs, and trapping EVs with porous structures. The device with antibody increases EV-capture efficiency. EVs have negatively charged phospholipid membranes and therefore move across filter membranes. Recent advancements in the use of exosomes as drug delivery systems. Kanchanapally, R. Drug-loaded exosomal preparations from different cell types exhibit distinctive loading capability, yield, and antitumor efficacies: a comparative analysis.

International Journal of Nanomedicine 14 , 1—10 Akers, J. Biogenesis of extracellular vesicles EV : exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. Muralidharan-Chari, V. ARF6-regulated shedding of tumor cell-derived plasma membrane microvesicles. Melo, S. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Kim, M. Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells.

Atha, D. Mechanism of precipitation of proteins by polyethylene glycols. Analysis in terms of excluded volume. J Biol Chem , — Abramowicz, A. Proteomic analysis of exosomal cargo: the challenge of high purity vesicle isolation. Keller, B. Interferences and contaminants encountered in modern mass spectrometry. Bhattacharjee, S. DLS and zeta potential - What they are and what they are not? Chang, M. Deregibus, M. Charge-based precipitation of extracellular vesicles.

Thery, C. Scheerlinck, E. Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry. Vergauwen, G. Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. Download references. We thank Prof. Lewis K. Pannell and Ms. Lindsay Schambeau for the mass spectrometry analyses of the exosomal preparations. Conception and design: A. Analysis and interpretation of data e. S and M. All authors read and approved the final manuscript. Correspondence to Ajay Pratap Singh. Reprints and Permissions.

Acta Tropica Biochimie Pharmaceutics Journal of Controlled Release Frontiers in Physiology By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content. Subjects Biological techniques Cancer. Abstract Exosomes have received significant attention for their role in pathobiological processes and are being explored as a tool for disease diagnosis and management.

Results Different exosome isolation methods yield different amount of exosomes To compare the isolation efficiency of different exosome isolation methods, we used culture supernatant from a pancreatic cancer line MiaPaCa which was available to us in abundant quantity. Figure 1. Full size image. Figure 2. Figure 3. Figure 4. Figure 5. Full size table. Figure 6. Figure 7.

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  • Discussion Exosomes are being widely appreciated for their role in cell-cell communication and as important mediators in multiple biological functions in carcinogenesis, immunosuppression, therapy resistance, etc. Table 2 Summery of different exosomes isolation methods. Protein-based exosomes quantification and immunoblot analysis Protein-based quantitation of isolated exosomes was done using the protein DC assay kit as described earlier by us 7 , Data Availability We confirm that all the data in this manuscript is original, stored with us and is available for sharing upon a reasonable request.

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