Isolation and Purification Services for Extracellular Vesicle

Communication between cells based on EVs (extracellular vesicles) is conserved in all life. Evidence shows that EVs have many advantages over traditional synthetic carriers, opening up new frontiers for modern drug delivery. Creative Biolabs offers research services related to the isolation and purification of EVs with specialized insights into the development and preparation of EVs.

EVs Isolation Approaches

EVs are lipid-structured vesicles secreted by cells and are widely found in cell culture supernatants as well as in various body fluids (blood, cerebrospinal fluid, saliva, urine, breast milk, etc.) EVs are to be separated from biospecimens so as to exclude background interference from other extraneous molecules. At Creative Biolabs, the EVs isolation platform supports the isolation of EVs from various sources, with available isolation methods including but not limited to:

Differential centrifugation: EVs would be separated by gradually increasing the centrifugal force or centrifugal time according to the differences in the settling coefficients of vesicles, cells, cell debris and protein molecules in solution. Differential centrifugation is a classical method for the isolation of EVs from various types of samples for functional studies and marker screening. The efficiency of differential centrifugation for the separation of EVs also depends on factors such as acceleration, rotor type, and viscosity of the sample. Therefore, the parameters are adjusted appropriately according to the sample type to obtain high-purity EVs.
Density gradient centrifugation: Based on the difference in density and size between vesicles and other biomolecules, EVs are separated from protein aggregates and non-membrane particles by adding separation media such as sucrose and iodixanol to stay in different gradient layers in solution. The method has high separation purity, and the buffering effect of separation media can also reduce the damage of EVs by centrifugal force, appropriate for functional studies of EVs, marker detection, and determination of contents.
Precipitation: Depending on the physicochemical properties of the compounds and EVs, EVs are isolated by co-precipitation or reverse screening, which mainly includes polymer precipitation and organic solvent precipitation. Polymer precipitation is a method that uses the hydrophobicity of membranes and associated water-soluble compounds to competitively bind water molecules to co-precipitate hydrophobic protein and lipid molecules, suitable for samples with RNA analysis. Organic solvent precipitation is a method that uses organic solvents that are miscible with water to significantly reduce protein solubility, resulting in precipitation and reverse separation of EVs.
Microfluidic: These include capture microfluidics, filtration microfluidics, magnetic separation microfluidics, acoustic separation microfluidics, and dielectrophoretic microfluidics. The microfluidics-based methods are emerging techniques for the separation and detection of EVs, and the advantages of automation make it extremely advantageous in the biological function study of EVs and clinical diagnosis of diseases.

Fig.1 Graphical summary of mainly used EV isolation methods. (Monguió-Tortajada, 2019)

EVs Purification Approaches

Purification of EVs facilitates the improvement of their quality and is a prerequisite for their characterization and further studies. Creative Biolabs offers diverse EVs purification strategies and services based on size, and immunological properties, including but not limited to:

Field-flow fractionation: This is one of the most commonly used particle size-based purification methods. The flowing fluid of the component to be separated is made to flow through a flat strip channel formed by upper and lower flat plates, and a field is applied vertically to the channel. The particles are subjected to both horizontal and vertical flow fields through the channel, with small EVs being subjected to less vertical force and spreading towards the channel center, while large EVs are subjected to more vertical force and moving closer to the accumulated wall, thus creating a size gradient in the vertical direction. The field will cause different components to be at different positions from the lower wall and thus move at different speeds to achieve purification and concentration of EVs. The flow can be passed through asymmetric fields such as electric, gravitational, thermal or semi-permeable membranes. The technique possesses low or no shear effect due to the absence of a stationary phase and the relatively low system pressure. Since no expensive equipment is required, it is suitable for large-scale purification and volume concentration of EVs.
Size-exclusion chromatography: A method for the purification of particles of different sizes based on the different velocities of the different particle sizes during the passage through the porous polymer gel packing. The purification of EVs is performed by gravity rather than centrifugal force, thus avoiding the aggregation of EVs caused by centrifugal force and the impairment of EVs membrane integrity and bioactivity. This method is suitable for functional studies of EVs, marker detection and analysis of contents.
Immunoaffinity: Based on the antigen-antibody immune reaction, the antibody specifically binds to the corresponding antigen on the surface of EVs to achieve the capture and purification of EVs. One of the most representative purification methods is the magnetic bead-based affinity method, whose main advantage is that magnetic beads provide a larger surface area for capturing EVs and improve purification efficiency. The ability to purify EVs of specific cellular origin makes it suitable for marker detection of EVs and clinical diagnostic studies.

Fig.2 Classification of the most common EV isolation methods by their purity, recovery and preferred use. (Clos-Sansalvador, 2022)

Creative Biolabs has accumulated insights into traditional and emerging methods for the isolation and purification of EVs, as well as proficiency in the suitable conditions and application prospects of each isolation approach. Our scientific team provides customized EVs isolation and purification services to advance EVs research. Please feel free to contact us to create a custom quote.

References

Monguió-Tortajada, M.;et al. Extracellular vesicle isolation methods: rising impact of size-exclusion chromatography. Cell Mol Life Sci. 2019, 76(12): 2369-2382.
Clos-Sansalvador, M.; et al. Commonly used methods for extracellular vesicles' enrichment: Implications in downstream analyses and use. Eur J Cell Biol. 2022, 101(3): 151227.

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