Exosome purification/isolation services
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 comparative table of the main methods for isolating extracellular vesicles, with their advantages and disadvantages (according EVerZom experience), followed by a summary of each method.
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. The final “concentrate” volume is limited by the instrument dead volume. TFF can also be used to purify some proteins from the sample according to the chosen cut-off, but cannot be considered as a complete purification method.
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. Ultracentrifugation is limited by a bad reproductibility, a low particle recovery from poorly concentrated solutions, and a fixed volume for each rotor, preventing scalability.
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 smaller molecules. However, SEC dilutes the particles, and requires a resin volume proportional with purified volume, which limits its scalability. New multimodal resins combine size-exclusion with binding properties, with the potential ability to solve SEC issues and be used in industrial production.
This method isolates particles based on their density. Swinging centrifuge tubes are loaded with an increasing density gradient from top to bottom, either with sucrose or iodixanol solutions. 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 for separating extracellular vesicles from protein aggregates or some lipoproteins. That is why this method is usually coupled with ultracentrifugation to increase the purity of a sample in academics. However, it suffers from the same drawbacks than UC: impossibility to scale-up, not reproducible yields. It is therefore reserved for research settings.
In this commonly used technique, EVs pass through a membrane with defined molecular weight cut-off, pushed by centrifugation force. Many academic laboratory have adopted this technique due to an easy and fast process. However, it is not adapted to industry, with a low recovery and purity, the impossibility to increase the scale, and the frequent membrane clogging. In addition, it appears difficult to find sterile ultrafiltration membrane.
Anion-exchange chromatography (AEX)
AEX is an innovative chromatography method in several steps: First the charged particles and molecules are captured by the resin with electrostatic interaction, and they are next released at the end by elution in a more concentrated salt solution. Theoritically, it offers a high capacity of both concentration and purification, together with a scale-up offered by GMP column commercially available. However, it requires some efforts to adjust the numerous parameters to allow good results.
Several extracellular vesicles markers have been identified and can be used to selectively isolate exosome such as tetraspanins CD9, CD63 and CD81. Antibodies against these extracellular vesicles proteins are commonly used in immunoaffinity capture. Magnetic beads or chromatography resin coated with these specific antibodies can capture positive extracellular vesicles, isolating them from other particles, before to release it during the elution phase with a salt solution. This would be potentially the best method in terms of selectivity. However, the lack of a specific marker express by all EVs prevents a perfect purification, and this technique is still limited to very small volumes of EVs.
To conclude, it appears that industrial process of EV downstream is currently driven towards a combination of TFF for EV concentration and a chromatography technique for EV purification from other biomolecules. The choice should be done depending on the target product (targeted purity, concentration…). Please contact EVerZom for any project related with EV isolation, we will have the solution for you.