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There are several methods to isolate extracellular vesicles from a fluid : some concentrate the sample in a smaller volume and some purify the fluid from other components. When a sample is concentrated, only the fluid is eliminated, most of the other particles remain (proteins, nucleic acids, etc.). A purification removes some of these particles. Removing proteins is important to reach a high ratio of extracellular vesicles particles to µg of protein : higher the ratio, higher the purity. (Watson et al.) The choice of the exosome isolation method will depend on several factors like the volume that will need to be purified, the final use of the exosomes (either for characterization or therapeutics purpose) and the targeted purity (it can differ from the field of therapeutic application : for instance in regenerative medicine, the whole secretome can be active and used as a whole).

Below is a summary of main extracellular vesicle isolation methods, how they compare and their advantages/disadvandages regarding concentration of EVs, free protein purification, recovery and scalability.

Ultracentrifugation

Ultracentrifugation can be used to isolate particles from a sample and concentrate them. They are separated according to their sedimentation coefficient which is mostly function of their size and density. A high centrifugal force is applied to a sample, sedimenting particles and separating them from the supernatant. To separate several types of particles from a sample according to their sedimentation coefficient, successive runs can be done. For extracellular vesicles, ultracentrifugation can be used to separate them from a sample and concentrate them in a smaller volume. It is the most commonly used method to isolate extracellular vesicles. This method also purifies extracellular vesicles from small proteins that remain in the supernatant, even though proteins aggregates can co-sediment with exosome, decreasing the purity of the sample.

Density gradient ultracentrifugation for colloidal nanostructures  separation and investigation - ScienceDirect
(Li et al. 2018)

Density gradient centrifugation

This method isolates particles based on their size and density. Swinging centrifuge tubes are loaded with an increasing density gradient from top to bottom. After the application of a centrifugal force, particles of same density end up at the same position in the gradient medium. Because density is the only factor, this method is efficient to purify extracellular vesicles from proteins, even aggregates. That is why this method is usually coupled with ultracentrifugation to increase the purity of a sample.

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(Li et al. 2018) ϱ = density of medium d = density of particles

Tangential Flow Filtration (TFF)​

Tangential Flow Filtration is a technique that separates particles from a liquid according to their size via a porous membrane. The flow parallel to the membrane prevents its blinding, letting liquid and some particles pass thanks to transmembrane pressure. The membrane is chosen according to its molecular weight cut-off, depending on which particles are intended to pass and whether the retentate or permeate is of interest. For extracellular vesicles, it is used to concentrate the retentate with cut-off smaller than extracellular vesicles molecular weight. The particles are then recovered by diafiltration without losing much of the product. TFF is an easy, fast and efficient technique for exosome concentration. It is scalable and therefore will be the method of choice for exosome concentration at large and clinical grade.
TFF can also be used to purify proteins from the sample according to the chosen cut-off.

What is Tangential Flow Filtration and how can it benefit your process? -

Size Exclusion Chromatography (SEC)

Size Exclusion Chromatography is a technique of proteins purification. It separates particles according to their size, using a column filled with a gel containing beads with pores of several size. As the sample goes through the column, small particles enter the pores of the beads, while bigger particles are not retained and elute earlier. Thus, as the elution goes, the size of particles that elute decreases. For extracellular vesicles, SEC is an easy way to purify them from proteins and nucleic acids while preserving their integrity and functionality.

Size exclusion chromatography - Labster Theory

Immunoaffinity capture

Several extracellular vesicles markers have been identified and can be used to selectively isolate exosome such as tetraspanins CD9, CD63 and CD81. Antibodies of these extracellular vesicles membrane surface proteins are commonly used in immunoaffinity capture. Magnetic beads coated with these specific antibodies binds to positive extracellular vesicles, isolating them from other particles. This is an efficient and easy method of isolation but also highly selective to a specific vesicle population that may not represent the initial sample. Moreover, easily removing the antibodies can be difficult and impact the integrity of exosome. Therefore, it can be very relevant for analytics and characterization purpose but hardly usable for therapeutics purposes.

Schematic of immunoaffinity-based exosome isolation. First, antibodies recognizing exosome-specific markers are immobilized onto solid matrices. After incubating exosome-containing fluids with antibody-conjugated solid matrices, exosomes can be enriched onto such solid matrices. Free exosomes can be collected via an additional elution step.
(Yang et al. 2020)

In addition to exosome characterization services, we provide exosome manufacturing, characterization and engineering services to our partners.

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