2.2. Size Based Techniques

Ultrafiltration is one of the most common size-based techniques used for exosome isolation; the idea behind this method is the same as with conventional membrane filtration, where the separation of particles is based on the size and molecular weight cut off (MWCO) of the membrane being used [104]. That is, particles larger than the MWCO of the particular filter are retained by the filter and particles smaller than the MWCO of the filter are passed through the filter into the filtrate [101,104]. One challenge with ultrafiltration is the clogging and trapping of vesicles (and therefore loss of exosomes) on the filter unit [116]. While this can be minimized by starting with larger MWCO filters and moving to smaller ones, it leads to low isolation efficiency, and the exosomes lost on the membrane cannot be used in downstream analysis [101]. While ultrafiltration is less time consuming than ultracentrifugation and requires no special instrumentation, it can still lead to particle deformation and lysis of exosomes due to the shear force, though this can be reduced by monitoring and regulating transmembrane pressure [106].

A commercially available isolation kit, the ExoMir Kit (Bioo Scientific; Austin, TX, USA) has been developed to isolate exosomes based on size. Essentially, two membranes (200 nm and 20 nm) are placed into a syringe with the 200 nm filter at the top and the 20 nm filter at the bottom. The sample is typically pretreated with a low speed centrifugation, to pellet cells and cellular debris, and proteinase K, to help breakdown larger particles and prevent the membrane from clogging. After pretreatment, the sample is passed through the syringe where the larger vesicles (>200 nm) remain above the first filter, the smaller vesicles (<200 nm and >20 nm) remain between the two filters in the syringe, and the smallest vesicles (<20 nm) are passed through the syringe and discarded. Other methods relying on the same general principle have also been developed, such as the ExoTIC technology, in order to make the isolation of exosomes a more clinically friendly procedure [117].

The idea behind sequential filtration for exosome isolation is similar to the ExoMir Kit or ExoTIC methods discussed in Section 2.2.2, in that it relies on a series of filtration steps for exosome enrichment. In sequential filtration, the initial steps involves filtration with a 100 nm filter to eliminate cells, cellular debris, and large rigid particles [118]. Particles that are larger than 100 nm in diameter, such as exosomes and microvesicles, are able to pass through the 100 nm filter as long as they are soft and flexible [118]. However, the more rigid components associated with cellular debris are filtered away [118]. The filtrate then undergoes tangential flow filtration with a 500 kDa MWCO membrane to remove soluble proteins and other contaminants [118]. Finally, concentrated retentate is then filtered with a 100 nm track-etch filter for exosome enrichment [118]. The primary advantages of this methodology are that it can isolate exosomes from 150 mL of media within a day, is automatable, and produces intact and biologically active exosome material, some of which have been used in clinical trials [118,119,120].

The use of size exclusion chromatography (SEC) to isolate exosomes from other EVs based on size is performed the same way as if one wanted to separate proteins of different sizes. That is, a column is packed with a porous stationary phase in which small particles can penetrate. This penetration slows down the movement of the smaller particles through the tube, causing them to elute later in the gradient, after the larger particles. Typically, size exclusion is used in parallel to ultracentrifugation methods, where the exosome pellet is re-suspended after enrichment by ultracentrifugation and then further purified using SEC [121,122]. While SEC methods preserve vesicle structure, integrity, and biological activity, they require run times of several hours, are not easily scalable, and cannot be used for high throughput applications [122]. However, iZON science has produced a qEV Exosome Isolation Kit, which allows for rapid, cost effective, high precision exosome isolation within 15 min based on the SEC methodology [123]. Their products allow for exosome isolation from <150 μL up to 10 mL volume of starting material with porous resins of 35 nm or 75 nm for optimal exosome isolation [123]. Development of such methodologies may bring about standardization in the area of exosome isolation, making the use of exosomes in the clinical setting more realistic.

Flow Field-Flow Fractionation (FFFF) is a new technique used to isolate exosomes based on size. In this method, the sample is injected into a chamber and subjected to parabolic flow as it is pushed down the length of the chamber [124]. At the same time, a crossflow (a flow perpendicular to the parabolic flow) is used to create the separation of the particles in the sample [124]. Larger particles are more affected by the crossflow, so they are pushed closer to the walls of the chamber, where the parabolic flow is slower [124]. Thus, the larger particles elute after the smaller particles, which are less affected by the crossflow, remain in the center of the parabolic flow, and elute earlier [124].

In traditional dialysis, separation of particles in the sample is achieved by diffusion of particles across a porous membrane. The selectivity of the separation is dependent on the MWCO of the given membrane; particles smaller than the MWCO of the membrane will diffuse across the membrane, and particles larger than the MWCO of the membrane will remain on the starting side of the membrane. In HFD, the sample is forced through a dialysis tube with a MWCO of 1000 kDa by hydrostatic pressure. The solvent and small solutes pass easily through the tube and the larger particles, such as exosomes and other EVs, remain in the tube, where they can be collected [125]. Typically, ultracentrifugation methods are used after HFD isolation to further separate exosomes from other EVs retained in the dialysis tube [125].