Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics

Abstract

Exosomes are small extracellular vesicles with diameters of 30-150 nm. In both physiological and pathological conditions, nearly all types of cells can release exosomes, which play important roles in cell communication and epigenetic regulation by transporting crucial protein and genetic materials such as miRNA, mRNA, and DNA. Consequently, exosome-based disease diagnosis and therapeutic methods have been intensively investigated. However, as in any natural science field, the in-depth investigation of exosomes relies heavily on technological advances. Historically, the two main technical hindrances that have restricted the basic and applied researches of exosomes include, first, how to simplify the extraction and improve the yield of exosomes and, second, how to effectively distinguish exosomes from other extracellular vesicles, especially functional microvesicles. Over the past few decades, although a standardized exosome isolation method has still not become available, a number of techniques have been established through exploration of the biochemical and physicochemical features of exosomes. In this work, by comprehensively analyzing the progresses in exosome separation strategies, we provide a panoramic view of current exosome isolation techniques, providing perspectives toward the development of novel approaches for high-efficient exosome isolation from various types of biological matrices. In addition, from the perspective of exosome-based diagnosis and therapeutics, we emphasize the issue of quantitative exosome and microvesicle separation.

Keywords: Exosome; diagnosis; extracellular vesicle; microfluidic; microvesicle; separation.

Figure 1

Schematic representation of differential ultracentrifugation-based exosome isolation. Differential ultracentrifugation is performed by multiple cycles of centrifugation with centrifugal forces from 300 ×g up to 100,000 ×g. After each centrifugation step, pellets including cells, cell debris as well as apoptotic bodies are removed while the supernatant was collected for further centrifugation. After the last centrifugation (i.e., 100,000 ×g), exosomes-containing pellets and contaminant proteins are collected by removing the supernatant. The centrifugation is performed at 4°C.

Figure 2

Schematic representative of gradient density ultracentrifugation-based exosome isolation. (A) Isopycnic density-gradient ultracentrifugation is prepared by adding medium in layers of progressively decreased density from bottom to top. After prolonged centrifugation, extracellular components including exosomes, apoptotic bodies and protein aggregates reach a static position in medium of similar density to each component. However, because isopycnic gradient ultracentrifugation depends solely on the density difference between different solutes in samples, this method cannot separate substances (e.g., microvesicles) with similar buoyant density to exosomes. (B) The moving-zone gradient ultracentrifugation normally consists two gradient medium sections. The top layer is a medium with density lower than all of the solutes of the sample. The bottom is a high-density cushion. As the density of the solutes are all greater than that of the gradient medium, after centrifugation, all solutes will be sequentially separated based on not only density, but also mass/size, thereby allowing the separation of vesicles of comparable density but varying size.

Figure 3

Schematic demonstration of ultrafiltration-based exosome separation. (A) Tandem- configured microfilter. Extracellular fluids are passed through tandem-configured microfilters with defined size-exclusion limits around 20-200 nm. When passing through the two membranes, large vesicles including cell debris, apoptotic bodies and the majority of microvesicles are trapped in the 200-nm membrane, while vesicles with diameter from 20 to 200 nm are retained on the lower 20 nm filter. (B) Sequential ultrafiltration. Extracellular fluids are first passed through a 1000-nm filter to get rid of larger particles (e.g., cells or cell debris); then the filtrate is passed through a second filter with 500-kD cut-off to remove small particles such as free proteins; finally, exosomes <200 nm are collected via a 200-nm filter.

Figure 4

Tangential flow filtration ensures highly efficient ultrafiltration. During tangential flow filtration, the feed stream flows parallel to the membrane face. The applied pressure causes one portion of the flow stream to pass through the membrane according to the filter size. As the membrane is constantly under a parallel flow force, potential clogging can be efficiently minimized. During the tangential flow filtration procedure, the remainder is re-circulated back to the feed reservoir for repeated filtration, ensuring thorough filtration.

Figure 5

Principle for Size-exclusion chromatography-based exosome isolation. When passing a solution through a stationary phase consisting of porous resin particles, molecules can be separated according to size (A); While particles with hydrodynamic radii smaller than that of the pores of the stationary phase enter into the pores for longer traffic distance, larger particles, which cannot enter the pores move directly around the resin (B). This causes particles with different sizes to exhibit different retention times and therefore facilitate size-based separation.

Figure 6

Schematic of Polymer Precipitation Strategy. After the addition of highly hydrophilic polymers to an exosome-containing solution, water molecules surrounding the exosomes are tied up by the polymers, lowering the solubility of the exosomes and inducing their subsequent precipitation. The exosomes can be easily collected with low-speed centrifugation.

Figure 7

Schematic of aqueous two-phase system-based exosome isolation. When the more hydrophobic polyethylene glycol (PEG) and more hydrophilic dextran solutions are mixed, a two-phase system could occur. After addition of PEG and dextran to exosome-containing solutions followed by incubation and low-speed centrifugation, proteins and other big molecular complexes preferentially accumulate into PEG while exosomes preferentially accumulate into the dextran phase.

Figure 8

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.

Figure 9

Aptamer-mediated immunoaffinity. Aptamers recognize and bind their target via conformational complementary. After adjusting key factors of the buffering system such as salt types and ionic strength, the shape of the aptamer undergoes change and releases the bound target molecules.

Figure 10

Integrated microfluidic technique allows combined exosome isolation and analysis. After adding exosome-containing fluids into the sheath medium, particles in the fluids including exosomes can be separated by different approaches based on the physical and biochemical properties of extracellular vesicles. Importantly, these miniaturized microfluidic apparatuses, facilitated by signal detecting platforms, allow for not only fast exosome isolation from small amount of body fluids, but also real-time exosome characterization for in situ diagnosis.

Figure 11

Principle of the nanowire-based exosome trip system. (A) Similar to SEC-based separation, a nanowire-on-micropillar hierarchy structure could be created via imprinting of porous silicon-consisting nanowires on the walls of the evenly separated micropillars. After adding exosome-containing fluids to the nanowire-on-micro-pillar tiered structure, particles in fluids are subject to different fates: (1). Larger particles (e.g., cell) are directly excluded from the sub-micrometer micropillar array; (2). Particles with submicron sizes (e.g., cell debris) are able to enter the micropillar interval but are unable to enter the 30-200 nm nanowire interval; (3). Small molecules (e.g., proteins) move across the nanowire interval without being obstructed; (4). Particles of 30-200 nm (e.g., exosomes) are arrested by the nanowire forest. (B) Particles with different sizes present different retention time and therefore facilitates size-dependent separation.

Figure 12

Contact-free microfluidic enables simplified exosome separation procedure. (A) In the viscoelastic medium flow-based microfluidic system, the exosome-containing fluids (added from inlet 1) meet the sheath flow (added from inlet 2) and are first aligned along the microchannel wall. After exertion of the elastic lift force that arises from viscoelasticity of the fluid, exosomes, and other extracellular components are driven toward the centreline of the microchannel according to their sizes, with larger particles eventually reach the centreline. (B) Under the pressure of ultrasound waves, particles with different mechanical properties (e.g., compressibility, size and density) experience differential radiation forces and results in contact-free and size-dependent exosome separation in a continuous manner.

Figure 13

Extracellular vesicles consist of mainly two types of vesicles with similar physiochemical properties. Extracellular vesicles include exosomes and microvesicles. The main differences between them lie in their subcellular origins. Microvesicles are 50-1000 nm shedding particles from cell membrane; exosomes are 30-150-nm extracellular vesicles originated from endosomes, they are secreted into body fluids through exocytosis after cell membrane and multivesicular body fusion. Due to a lack of effective strategy to separate microvesicle and exosome, it is still difficult to precisely assess their physiochemical properties and functions.

Figure 14

Calculation of the proportion of exosome and microvesicle components of extracellular vesicles via exosome and microvesicle marker-specific antibodies. The extracellular vesicles A (A') were concentrated via polymer precipitation. Then exosomes (B) or microvesicles (B') were extracted using corresponding antibody-based immunoaffinity capture; after elution, exosomes (C') and microvesicles (C) were extracted again from the elutes using antibody-based immunoaffinity method. Then, the extracted exosomes (B, C') and microvesicles (B', C) were quantified. The proportion of exosome and microvesicle in the original extracellular vesicles was calculated using the formulation as shown in D.