Exosomes are 30 to 150 nm diameter membranous vesicles of endocytic origin that are released by cells into the extracellular space, and contain various important biological molecules, such as lipids, proteins, and genetic materials. Exosomes have been found to participate in cell–cell communication, cell maintenance, some disease progression, and immune responses. Analysis of exosomes is of great significance in studying metabolic diseases, tumor metastasis, immune regulation, and so forth. In addition, exosomes have the ability to act carriers of biomarkers for clinical diagnosis and are ideal for the development of drug delivery systems.
Highly efficient methods for exosome isolation are prerequisites to obtain substantial breakthroughs. A large number of methods based on isolation by ultracentrifugation, immunoaffinity capture, size of exosomes, and polymer precipitation have been developed for isolation of exosomes.
Ultracentrifugation
With the capacity to generate centrifugal forces as high as 1,000,000 ×g, ultracentrifugation (UC) is an optimal process for separating small particles, such as bacteria, viruses, and cellular organelles. Differential UC is the most widely used method for exosome isolation, and is typically regarded as the gold standard, because it requires little technical expertise, little to no sample pretreatment, and affordability.
Density gradient centrifugation is another commonly used UC method that is very effective in separating exosomes and other extracellular vesicles (EVs), from protein aggregates and non-membranous particles. Density gradient centrifugation is particularly useful for separating exosomes and other EVs from bodily fluids. However, like differential UC, recovery of exosomes using density gradient centrifugation is low.
Size Exclusion Chromatography
The specific size of exosomes has been well utilized for purification. Based on this mechanism, various methods have been applied for exosome isolation, such as ultrafiltration, hydrostatic filtration dialysis, flow field-flow fractionation (FFFF), and size exclusion chromatography (SEC).
SEC is a technique for the isolation of exosomes from other EVs with a porous stationary phase that allows the separation of different molecules according to their sizes. The material that packed in a SEC column is composed of small particles of silica or polymer that have pores in which the molecules are trapped, so larger molecules are retained and elute quickly, while smaller molecules can penetrate them and be retained for longer.
The main advantages of SEC for exosome isolation are simple and time-consuming, allowing efficient removal of contaminated proteins and separation of exosome subtypes, and high purity of isolated EVs. Typically, exosome pellets are re-suspended after enrichment by UC and then further purified using SEC. Although the SEC method preserves the structure, integrity, and biological activity of exosomes, it cannot be used for high-throughput applications because large sample loading volume result in inefficient separation.
Products
SEC is the most efficient method for isolating EVs from biological liquids in just one step, with a high recovery rate and almost complete removal of contaminants. Amerigo Scientific offers 70nm and 35nm SEC columns for the isolation of EVs from complex biological fluids such as plasma, serum, urine, and cerebrospinal fluid.
Benefits
- Time saving: easy and rapid
- High purity: protein removal and high-density lipoprotein (HDL) purification
- Excellent recovery: method that maximizes recovery (>50%)
- No aggregation: method that reduces the risk of protein complex formation and vesicle aggregation
- Indicated for low volume
- Reusable columns and efficient packaging
- Protocol compatible with RNAs extraction and RT-PCR
- Successfully separating EVs of different sizes
Elution Profile
Amount of EVs and protein in each fraction from the column. Comparative of protein (BCA) vs vesicles (FACS CD63+/CD9+) content. SEC fractions were loaded on SD-PAGE and immunoblotted for CD9 tetraspanin with anti-CD9 (VJ1/20), under no-reducing conditions.
Polymer Precipitation Solutions
Polymer-induced precipitation is a common strategy for exosome separation, which is based on the interaction between highly hydrophilic polymers and water molecules around the exosomes to form a hydrophobic microenvironment, leading to exosome precipitation. Among various hydrophilic polymers, polyethylene glycol (PEG) as a non-toxic polymer has been commonly used.
The polymer precipitation method is simple, fast, easily scalable and can provide high yields of EVs with a well-kept structure. However, interference by co-precipitated contaminants is inevitable because of the nonspecific interaction between hydrophilic polymers and various water-soluble substances (nucleic acids, lipoproteins, proteins, etc.), which might be solved by the integration of other separation methods.
Products
Amerigo Scientific offers easy and rapid precipitation solutions to increase the sensitivity of biomarker detection.
Benefits
- Easy and rapid precipitation solution
- Ultracentrifugation free method for the highly- sensitive biomarker detection
- Very clean and better exosomes preparation, reducing carry-over albumins and immunoglobulins compared to other methods
- Isolated intact exosome suitable for a great variety of protein-sensitive applications and downstream uses
Immunoaffinity Capture-based Isolation
A number of specific membrane proteins are expressed on the surface of the exosome, such as CD9, CD63, CD81, CD82, programmed cell death 6 interacting protein (also known as ALIX), annexin, epithelial cellular adhesion molecule (EPCAM), and RAB5, which can be used as specific markers for the isolation of exosomes. Immunoaffinity capture-based techniques utilize antibodies to capture exosomes by targeting specific antigens on the surface of the exosome. These antibodies can be attached to plate (such as ELISA), magnetic beads, resins, and microfluidic devices for use.
Immunoaffinity methods can be used to isolate a subpopulation of exosomes from complex mixtures and can also be used to separate exosomes from other types of EVs. Immunoaffinity method provides lower yield of isolated exosomes with higher purity than methods that isolate exosomes based on other properties. The main limitation of this method is that the antigens used to capture exosomes must be expressed on the exosome surface, since antibodies are not able to capture antigens within vesicles. Due to the complexity of the composition of biological fluids, immunoaffinity techniques are often used after enrichment of exosomes by ultracentrifugation or ultrafiltration.
Products
Amerigo Scientific offers a wide range of exosome capture beads to isolate specific exosomes from biological fluids without previous enrichment procedures.
Benefits
- Allowing the detection of isolated exosomes from different isolation techniques, such us differential ultracentrifugation, precipitation solutions or size exclusion chromatography columns.
- Direct exosome isolation without previous enrichment
- Fully compatible with downstream analysis (WB, mRNA, miRNA, etc.)
Exosomes are difficult to identify because of their small size, strong heterogeneity, and low refractive index. The use of flow cytometry not only enables high-throughput analysis of exosomes, but also allows quantification or classification of exosomes based on their antigenic expression.
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