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Exosomes are nano-sized biovesicles released into surrounding body fluids after the fusion of multivesicular bodies with the plasma membrane. They carry cell-specific cargos of proteins,
lipids, and genetic materials, and can selectively be taken up by neighboring or distant cells reprogramming the recipient cells’ biology[1]. They play a role in the body’s physiological
and pathological processes and have been discovered in almost all body fluids, including blood, urine, saliva, breast milk, cerebrospinal fluid, semen, amniotic fluid, and ascites.
Figure 1. Classification of extracellular vesicles[2]
Exosomes are the best defined extracellular vesicles (EVs) so far and their biogenesis, release pathways, size, content, and function are different from microvesicles and apoptotic bodies[3].
Feature
Exosomes
Microvesicles
Apoptotic bodies
Size
40-150 nm
150-1000 nm
﹥1000 nm
Density
1.11-1.19 g/mL
1.02-1.22 g/mL
1.16-1.28 g/mL
Origin
Multivesicular bodies fusion with plasmatic membrane
Direct outward budding or blebbing from the plasma membrane
Cellular debris, plasma membrane blebbing during cell apoptosis
Markers
Alix, TSG101, HSC70, CD63, CD81, CD9, flotillin-1
Selectins, integrins, CD40, MMPs， Annexin A1
Annexin V, DNA, histones
Membrane permeability
Membrane impermeable
(PI negative)
Membrane impermeable
(PI negative)
Membrane permeable
(PI positive)
Functions
Intercellular communication
Intercellular communication
Physiological and pathological regulation
Table 1. Characteristics of exosomes, microvesicles, and apoptotic bodies
Exosomes were first discovered in the cultured supernatant of sheep erythrocytes in 1983. Three pioneer scientists won the Nobel Prize in Physiology or Medicine in 2013 for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells.
Recently, exosomes received a lot of attention due to their emerging role as intercellular messengers and their potential in disease diagnosis. Exosomes contain proteins, lipids, and RNAs that are specific to their cell origin and can deliver cargo to both nearby and distant cells[4]. Since exosomes are easy to purify/extract and detect, hence offering opportunities for disease diagnosis and treatment.
Engineering transformation and modification of exosomes can optimize their natural targeting and delivery to a specific location, hence broadening their use in clinical treatment with low immunogenicity, high biocompatibility and high efficacy.
A recent study in Nature Biomedical Engineering reported a novel inhalable SARS-CoV-2 vaccine candidate consisting of a recombinant SARS-CoV-2 receptor-binding domain (RBD) conjugated to lung-derived exosomes. Since lung spheroid cells- (LSCs)derived exosomes (LSC-Exo) share surface proteins and receptors with membrane features found among airway epithelia, hence these exosomes are more distributed and retained for longer time in both the mucus-lined respiratory airway and in lung parenchyma and have enhanced internalization by antigen presenting cells (APCs) in the lung, providing a more targeted delivery carrier than commonly used liposomes. In hamsters, two doses of the vaccine attenuated severe pneumonia and reduced inflammatory infiltrates after exposure to live SARS-CoV-2[5].
Figure 2. Schematic representation of the fabrication of the RBD-Exo vaccine
The vaccine is delivered into the lungs via inhalation. RBD-Exo induces mucosal immunity and systemic immunity with the generation of RBD-specific IgA and IgG antibodies against SARS-CoV-2 infection in hamsters.
Isolation and purification of Exosomes
Cumulative evidence has revealed that exosomes can play an exceptional role in diagnostics and therapeutics of multiple diseases. An efficient, simple, and affordable method to isolate intact and pure exosomes is crucial to carrying out relevant pathways or disease research.
Herein, we summarized the commonly used exosome isolation techniques.
Strategy
Principle
Advantages
Disadvantages
Density gradient centrifugation
Components with imparity of size and density Possess various sediment speed
High purity, avoiding exosomal damage
Labor-intensive, Preliminary preparation and Cumbersome operation
Ultrafiltration
Particles with various size and molecular weight
Convenient, Without special equipment and reagents
Clogging on filtering membrane,
Loss of exosomes of small particle diameter
Immunoaffinity
Based on interaction between antibodies and specific membrane proteins of exosomes
High specificity for exosome subtypes isolation
Expensive, depending on specificity of the antibody
Polymer precipitation
The influence of exosomal solubility or dispersibility under the high hydrophilic polymers
Imple operation, suitable for large-volume samples
Potential contaminants (co-purifying protein aggregates or residuary polymers)
Size exclusion chromatography (SEC)
Particles with various size and molecular weight
Simple, economical, maintain the biological function and structure
Special columns and packing are required, lipoprotein contamination
Table 2. Comparison of exosome isolation techniques[6]
Furthermore, highly pure exosomes can be easily obtained with exosome isolation kit
Figure 3. Exosome isolation procedures
Labeling and Identification of Exosomes
In 2014，the International Society for Extracellular Vesicles (ISEV) provided researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or function to extracellular vesicles (EVs). ISEV suggests a format of characteristics of EVs that should be analyzed and at least 2 different technologies should be used to characterize individual EVs.
Detection of exosome-enriched proteins
Analytic techniques to detect exosome-enriched proteins include Western blot (WB), high resolution flow cytometry (FACS) or global proteomic analysis using mass spectrometry to identify exosome transporting-related transmembrane proteins (CD63/CD81/CD9), heat shock proteins (HSP60/HSP70/HSPA5/CCT2/HSP90) and some cell-specific proteins that are enriched in exosomes membrane. Among them, CD63 and TSG101 are the most commonly used exosome markers.
Identification of exosome morphology with TEM
Exosomes morphology can be directly identified under transmission electron microscopy (TEM) with high resolution to provide an indication of the heterogeneity of the samples.
Size distribution and concentration measurements with NTA
NTA (Nanoparticle Tracking Analysis) technology can quickly and accurately analyze the particle size distribution and concentration of large number of exosomes in the sample.
A study by Muyu Yu et al. focused on whether exosomes derived from the bone marrow MSC (BMSC) pretreated with atorvastatin (ATV) could exhibit better pro-angiogenic ability. As shown in Fig 4, they isolated exosomes from non-pretreated BMSC (Exos) and ATV pretreated BMSC (ATV-Exos) and evaluated their characterization by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting[7].
Figure 4. The characterization of BMSC-derived exosomes[7]
a. The morphology of Exos and ATV-Exos was examined by TEM. b. The specific surface markers (Alix, TSG101, CD81) of Exos and ATV-Exos were assessed by Western blotting. c. The diameter and particle concentration of Exos and ATV-Exos were detected via NTA.
Labeling and tracing of exosomes
In vitro labeling or in vivo tracing of isolated exosomes is helpful for further studies on the functions and applications of exosomes. There are many methods for labeling and tracing of exosomes, mainly including lipophilic tracers and membrane-permeable compound tracers.
Lipophilic Tracers
Lipophilic dyes (DiO, DiI, DiD, DiR) can label exosomes well, and are also commonly used dyes in exosome research.
In vitro tracing: Exosomes can be traced in vitro by using PKH26 dye and scanning it by confocal microscope. As shown in Fig. 5, PKH26-labeled exosomes (red) were localized in the perinuclear region of HUVEC by using laser scanning confocal microscopy, confirming the internalization of exosomes by endothelial cells[7].
Figure 5. The uptake verification of Exos and ATV-Exos by HUVEC[7]
Exosomes, cytoskeleton, and cell nucleus were stained red, green, and blue, respectively
In vivo tracing:
In vivo tracing can be done by using DIR dye and scanning by IVIS 200 Optical Imaging System.
Figure 6. In vivo imaging of DIR-labeled 4T1 exosomes in mouse mammary fat pads[8]
Intravenous injection of 4T1 exosomes into Balb/c mice with 4T1 tumors. Exosomes are labeled with the lipophilic fluorescent tracer DIR. A scale of the radiance efficiency is presented to the right of each live mouse image
Membrane permeable compound tracers
The membrane-permeable carboxyfluorescein diacetate succimidyl ester (CFSE) is a lipophilic fluorescent dye that can passively diffuses into cells, which is also commonly used for effective tracing of exosomes. It is colorless and non-fluorescent until the acetate groups are cleaved by intracellular esterases to yield highly fluorescent carboxyfluorescein succinimidyl ester. The succinimidyl ester group reacts with intracellular amines forming fluorescent conjugates that are well retained by cells.
Figure 7. Confocal images of CFSE-labeled embryonic tissue exosomes[9]
MCE has launched multiple exosome-related products. Please check our website for specific product information.
All MCE products are only used for scientific researches or drug registration applications, we do not provide products and services for any personal use.
