Exosome Isolation and Detection

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Exosome Isolation and Detection

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Isolation and Detection of Extracellular Vesicles


percipitation ultrafiltration size exclusion chromotography
ultracentrifugation density gradient centrifugation immunoaffinity

It is well established that extracellular vesicles (EVs) are important mediators of intercellular communication in health and disease. However, the specific contribution of EVs compared to other extracellular messengers can be difficult to elucidate. The first step of determining the precise role of EVs is to isolate them from biofluids or cell culture media.

There are many methods available to isolate EVs and each researcher must determine which method is best suited to their research question. Is the goal to determine a specific biomarker for cancer carried by EVs? In this case, the isolation method may focus more on quantity, rather than specificity, of EVs. Is the goal to determine what cargo is carried by EVs secreted from a particular cell type? Then perhaps, a more targeted isolation approach is required.


How do you isolate exosomes?

The chosen method of isolation determines the yield and relative “specificity” of an EV preparation. Traditionally, exosome isolation has been a time consuming and arduous task with many centrifugation steps at extremely high speeds. Other methods have been developed that have a simpler workflow and a faster turnaround time but may result in isolation of both exosomes and microvesicles. There are advantages and challenges associated with each technique, and each researcher must decide based on their individual system and ultimate goals which technique is optimal for them.

Schematic showing the key players of the ENS with the neuronal connections innervating the intestinal layers, the metabolites that are circulated to the CNS, and the proteins expressed by the vagus nerve and spinal cord.

In addition to the challenges associated with each technique, individual biofluids present complications based on their composition. For example, biological fluids such as plasma and serum are extremely complex and have higher viscosity than cell culture medium. Often, isolation of EVs from these biofluids requires faster and longer ultracentrifugation steps than when cell culture media is the source. Additionally, urine is an extremely useful source of EVs from the kidneys, bladder, and prostate, but the abundance of Tamm-Horsfall protein (THP) in urine can obscure detection of subtle, but important, changes in EV composition and protein expression. It is highly recommended to remove THP with a salt precipitation step prior to EV isolation.


Biofluid

Possible Contaminants/Co-isolated with EVs

Primary EV Source

Plasma
  • Proteins
  • Lipoproteins
  • Protein Aggregates
  • Erythrocytes
  • Platelets
  • NK Cells
Urine
  • Tamm-Horsfall Protein (THP)
  • Salt
  • Urea
  • Metabolites
  • Kidneys
  • Bladder
  • Prostate
Cerebrospinal Fluid (CSF)
  • Enzymes
  • Neurotransmitters
  • Ions (Na+, Mg2+, Cl-)
  • Neurons
  • Gila




Common Methods of EV Isolation

Precipitation

Yield: Intermediate Specificity: Intermediate (Varies)

Of the common techniques used to isolate EVs, precipitation offers one of the fastest turnarounds and most straightforward workflows. Briefly, samples containing EVs are incubated with a buffer that changes their solubility or sedimentation rate. One common precipitation buffer uses polyethylene glycol (PEG), a hydrophilic polymer. By incubating EVs with PEG, it decreases the solubility of EVs in solution and, using a very gentle centrifugation step, causes accumulation of EVs at the bottom of the tube. Some other common methods of precipitation include protamine, sodium acetate, and organic solvents. Precipitation results in a large volume of isolated EVs with no specificity for EV subtype or specific proteins.


Advantages

Disadvantages

Precipitation
  • High yield
  • Process many samples
  • Simple procedure
  • No specialized equipment
  • Maintain vesicle morphology
  • Difficulty separating EVs from protein aggregates, lipoproteins, and viruses
  • Possible contamination with precipitating polymer or chemical


Product

Specifications

EV Precipitation Solution (Blood)

For precipitation of EVs from small volumes (100-250 µL) of plasma and serum

EV Precipitation Solution (Blood)

For precipitation of EVs from up to 5 mL of cell culture supernatant

EV Precipitation Solution (Urine)

For precipitation of EVs from up to 20 mL of urine


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Ultrafiltration

Yield: Intermediate Specificity: Intermediate (Varies)


Illustration of ultrafiltration techniques using tangential flow filtration and traditional diafiltration.

Ultrafiltration is the process the fluid passing over a semi-permeable membrane, allowing some substances to pass through while others are retained. In the case of EV isolation, ultrafiltration involves passing your sample over a porous membrane filter between 0.08-0.1 µm in diameter. Due to the semi-permeable nature of the membrane, ultrafiltration will concentrate the sample, as well as separate molecules based on size. This technique is especially useful for isolating EVs from dilute biofluids, such as urine, as well as cell culture media. Broadly, there are two types of ultrafiltration commonly used for EV isolation: tangential flow filtration (TFF) and diafiltration. TFF passes the liquid horizontally (tangentially) over the pores of the membrane allowing particles that are smaller than the membrane pores to filter through, while larger particles are retained in the chamber. Diafiltration involves pushing the mixture directly up or down through the membrane pores.


Advantages

Disadvantages

Ultrafiltration
Diafiltration and Tangential Flow Filtration (TFF)

  • Protocol is simple and straightforward
  • Pure EV preparations
  • Concurrent processing of many samples
  • Pure EV preparations
  • No limit of sample volume
  • Concentration of dilute samples (urine, cell culture media)
  • Reduced filter clogging (TFF)
  • Some loss of sample in filtration
  • Clogging of filter (Diafiltration)
  • Some loss of vesicle morphology (Diafiltration)
  • Contamination with protein aggregates


Product

Specifications

Tangential Flow Filter (TFF)- EV Concentrator

For concentration of dilute matrices like urine and cell culture medium prior to EV isolation.


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Size-Exclusion Chromatography (SEC)

Yield: Intermediate Specificity: Intermediate


Illustration of exosome isolation by size exclusion chromatography. Image shows sample loading and different elution fractions for exosomes and circulating proteins.

As indicated by the name, size-exclusion chromatography (SEC) separates macromolecules based on their size. To accomplish this, the sample is passed through a porous column and molecules are isolated in fractions from the eluate. The size of the pores can vary depending on the size of the desired product. For isolation of EVs from proteins and lipoproteins, pore sizes can vary from ~40-80 nm. As the sample passes through the column, molecules smaller than the pore size will get trapped in the pores, slowing them down, while larger molecules will pass through. This results in the larger molecules, such as EVs, eluting from the column in earlier fractions, while smaller molecules, such as proteins and protein aggregates, are eluted in later fractions. To optimally use SEC for dilute matrices, such as urine or cell culture media, first these samples must be concentrated by an ultracentrifugation or ultrafiltration step.



Advantages

Disadvantages

Size-exclusion Chromatography

  • Reproducible
  • Preserves vesicle integrity
  • High sample recovery
  • Easily scalable
  • Limits EV aggregates
  • Extremely customizable (size of pore, volume of column, length of column)
  • Restricted sample volume
  • Low resolution of similarly sized molecules (must be at least 10% different to resolve)
  • Only one sample processed at a time


Product

Specifications

EV SEC Columns

To isolate EVs from ~2 mL plasma or concentrated conditioned media and urine.

EV MaxiSEC Columns

To isolate EVs from ~20 mL concentrated conditioned media


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Ultracentrifugation (UC), with differential centrifugation (DC)

Yield: Intermediate Specificity: Intermediate


Illustration of example ultracentrifugation protocol for exosome isolation.

Protocol adapted from Doyle LM, Wang MZ. Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis. Cells. 2019;8(7):727. Published 2019 Jul 15. doi:10.3390/cells8070727. *Optional step.


Considered the gold standard for EV isolation, ultracentrifugation with differential centrifugation relies on vesicle size and density. Briefly, samples are taken through several cycles of centrifugation to clear the supernatant of cells, cellular debris, and larger vesicles such as apoptotic bodies. Then the sample is subjected to either one or two rounds of ultracentrifugation (100,000-200,000 g). Some protocols suggest an additional filtration step using a 0.45-0.22 µm filter prior to ultracentrifugation.



Advantages

Disadvantages

Ultracentrifugation

  • Isolation from large volumes
  • No added chemicals
  • Equipment availability
  • Low RNA yield
  • Damage to exosomes at high speeds
  • Efficiency depends on rotor
  • Sample viscosity impacts sedimentation
  • Hard to scale up (limited by number of samples in ultracentrifuge run)

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Density gradient centrifugation

Yield: Low Specificity: High


Illustration of continuous density ultracentrifugation technique for exosome isolation. Illustration of stepwise density gradient centrifugation technique for exosome isolation.

To further improve upon the specificity of EV isolation, many researchers use density gradient centrifugation. There are two general types of density gradient centrifugation for EV isolation: continuous and stepwise gradients. Often called a “cushion”, continuous density gradient centrifugation involves underlaying a sucrose gradient of a singular density to the sample prior to ultracentrifugation. For stepwise, or discrete, density gradient centrifugation, sample is overlayed on layers of iodixanol with different densities.



Advantages

Disadvantages

Density Gradient Centrifugation
(Sucrose or Idoixanol)

  • Very pure populations (Iodixanol can separate EVs from virions)
  • Intact vesicle morphology
  • Requires ultracentrifuge
  • Time consuming (up to 2 days)
  • Hard to scale up (Limited by number of samples in ultracentrifuge run)

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Immunoaffinity capture

Yield: Low Specificity: High


Close up view of one well of 96-well plate showing exosome binding to antibody coated well. Close up view of immunocapture bead coated with antibody for exosome isolation.

As more cell-type and disease-specific proteins are identified, more researchers are turning to immunoaffinity capture techniques for EV isolation. This technique relies on the antigen-antibody interaction. Antibodies directed at target of interest (eg CD9, CD63) are coated either in a 96-well plate or conjugated to a bead. Sample is applied directly into coated well, or into sample with coated beads, and EVs are allowed to bind. In a final step, EVs can be eluted from the antibody and used in downstream functional and phenotypic analysis. This method of isolation is highly specific, resulting in extremely pure EV populations.



Advantages

Disadvantages

Immunoaffinity capture

  • High purity
  • High selectivity
  • Lack of specific markers for isolation
  • Difficulty separating EVs from antibodies or beads
  • Sample volume


Product

Specifications

CD9 Immunobeads for Exosome Isolation

Coated with anti-human CD9 for isolation of exosomes from plasma, serum, and concentrated urine.

CD63 Immunobeads for Exosome Isolation

Coated with anti-human CD63 for isolation of exosomes from conditioned cell media.


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How do you detect exosomes?

There are many methods available to confirm the presence of EVs after isolation. No matter the chosen method of detection, it is important to compare the isolated EV preparation to a known sample containing EVs of interest. Bio-Techne offers lyophilized exosome standards from a range of cell types, disease states, and biological fluids to serve as a positive control in detection assays.


Method

Advantages

Disadvantages

Electron Microscopy

  • Determination of size of vesicles (nm resolution)
  • Discrimination between EVs and other small particles (proteins, lipoproteins)
  • Visualize biogenesis
  • Preparation can result in loss of vesicle shape (cup-shaped)
  • Specialized equipment
  • Time intensive
  • Cost

Western Blot

  • Cost
  • Well-established workflow
  • No information about co-expression of markers
  • Large sample volume required

Simple Western

  • Automated
  • Well-established workflow
  • Small sample volume
  • No information about co-expression of markers

ELISA

  • Highly customizable
  • When combined with exosome standards, can be used for quantification
  • Small sample volume
  • Limited in scope to maximum expression of two protein markers (capture and detection antibodies)
  • Analysis of bulk populations of exosomes

Flow Cytometry

  • Single vesicle resolution
  • Co-expression of multiple protein markers
  • Cytometers must be specifically calibrated for EVs
  • Careful filtration of all reagents to minimize debris


Western blot of mesenchymal stem cell (MSC)-EV precipitation from MSCs culture media was verified by western blot. Blots were probed with Mouse Anti-Human CD9 (5G6) (NBP2–22187) and Mouse Anti-Human CD81 (1D6) (NB100-65805). Input, media prior to ultracentrifugation. Output, supernatant after ultracentrifugation. Pellet, EV pellet after ultra-centrifugation. NC, negative control. N-EV, EV from naïve MSCs. H-EV, EV from hypoxia challenged MSCs. Image was taken from Ren W, Hou J, Yang C, et al. Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promote non-small cell lung cancer cell growth and mobility as well as macrophage M2 polarization via miR-21-5p delivery. J Exp Clin Cancer Res. 2019;38(1):62. Published 2019 Feb 8. doi:10.1186/s13046-019-1027-0. Licensed under CC license.

Western blot showing detection of exosome markers CD9 and CD81 in exosome pellet, not in supernatant after ultracentrifugation.

Western Blot Exosome Marker Antibody Pack


Microscopy image with fluorescent tags showing expression of CD9, CD81, and CD63 on extracellular vesicles from two cell lines.

Simple Western lane view of conditioned media (CM) and exosomal fraction (Ex) of human colorectal adenocarcinoma cell line HT-29. Exosomal fraction was isolated using precipitation method. Using polyclonal Goat Anti-Human EpCAM/TROP1(AF960) a strong signal is detected in the Ex, but not CM, lane.


Simple Western Exosome Marker Antibody Pack


Vesicle Flow Cytometry

Flow cytometry is a powerful tool that allows for detection and characterization of multiple protein markers, both intracellularly and on the surface of many cell types, at single-cell resolution. This technology can also be applied to EVs, providing valuable information about vesicle phenotype and insights into probable function. However, there are many important considerations when using flow cytometry for the study of EVs.


  1. All fluids should be filtered through a 0.03 µm pore (including staining buffers and sheath fluid) to better discriminate EVs from debris.
  2. Traditional flow cytometers with FSC/SSC firing from the blue (488 nm) laser are not sensitive enough to detect nanometer-sized particles by light-scatter.
    1. Cytometers that can fire SSC from the violet laser (405 nm) are more sensitive for EV-based flow.
    2. Acoustic-wave cytometers can offer enhanced resolution compared to traditional hydrodynamic cytometers.
    3. Membrane-based dyes allow for use of fluorescence to detect EVs by flow.
  3. Instrument calibration is critical.
    1. For detection with light-scatter, use beads ranging from 80-1300 nm to confidently discriminate EVs from instrument noise.
  4. Due to their small size, fluorescence intensity from an EV will be dimmer than fluorescence intensity on a cell, so voltage adjustments must be considered.
    1. Novus Biologicals offers fluorescent exosome standards and Max Rainbow Fluorescent Particles to help calibrate instruments for vesicle cytometry using fluorescence.
    2. Use very bright fluorescent dyes for conjugated antibodies including PE, PE/Atto594, AlexFluor® 647, and APC.

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Select References

Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 2018;7:. https://doi.org/10.1080/20013078.2018.1535750.

Doyle L, Wang M. Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis. Cells 2019;8:727. https://doi.org/10.3390/cells8070727.

Crescitelli R, Lässer C, Lötvall J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat Protoc 2021;16:1548–80. https://doi.org/10.1038/s41596-020-00466-1.

Konoshenko MY, Lekchnov EA, Vlassov A V., Laktionov PP. Isolation of Extracellular Vesicles: General Methodologies and Latest Trends. Biomed Res Int 2018. https://doi.org/10.1155/2018/8545347.

Cocozza F, Grisard E, Martin-Jaular L, Mathieu M, Théry C. SnapShot: Extracellular Vesicles. Cell 2020;182:262-262.e1. https://doi.org/10.1016/j.cell.2020.04.054.