Exosomes are extracellular vesicles, generally sized between 30 and 150 nanometers, released by most cell types. They function as messengers, carrying proteins, lipids, and nucleic acids between cells, playing a significant role in intercellular communication. To study these vesicles, researchers must first separate them from the surrounding biological fluid, a process that is often complex and challenging. Biological fluids are complex mixtures containing soluble proteins, lipoproteins, and cellular debris. Purification is necessary to isolate a clean population of exosomes and ensure experimental results reflect exosome function rather than contamination.
Biological Sources Requiring Isolation
Exosomes can be isolated from virtually any biological fluid, and the choice of starting material dictates the necessary purification strategy. Common sources include cell culture media, which offers a controlled environment and relatively lower levels of initial contamination. This controlled environment simplifies the downstream isolation process compared to complex clinical samples.
Clinically relevant samples often include blood plasma or serum, urine, saliva, and cerebrospinal fluid. The specific source material dictates the initial level and type of contaminating molecules present. For instance, plasma contains high levels of lipoproteins and abundant soluble proteins that must be removed. Urine presents challenges, requiring large initial volumes and often containing crystalline structures and cellular debris. Therefore, the purification strategy must be tailored to the specific contaminants found in the starting material to achieve sufficient purity.
Physical Methods for Exosome Separation
Physical methods for exosome separation rely on differences in physical properties like size, mass, and density. Differential ultracentrifugation is a classic, though laborious, method that separates particles based on their mass and sedimentation rate. Samples are spun at increasing speeds, typically reaching up to 100,000 times the force of gravity (x g). Larger, denser contaminants pellet first, leaving exosomes in the supernatant until the final high-speed spin.
A more refined version, density gradient ultracentrifugation, further separates vesicles by layering the sample over a medium like sucrose or iodixanol. Exosomes migrate to the specific layer matching their buoyant density, typically between 1.10 and 1.21 grams per milliliter, providing a purer separation than differential spins alone. While ultracentrifugation yields large quantities, the prolonged high-speed forces can potentially lead to vesicle aggregation or structural damage.
Size Exclusion Chromatography (SEC) separates exosomes based purely on their hydrodynamic size using a packed column of porous beads. The column material, often cross-linked agarose, contains pores that dictate the path of the solution. Larger components, such as cellular debris and aggregated proteins, are excluded from the pores and elute quickly. Exosomes enter the pores and are temporarily retained, leading to their elution after the larger contaminants but before small soluble proteins. SEC is highly reproducible and gentler on the vesicles than ultracentrifugation, often resulting in a purer, more functional exosome sample.
Chemical and Affinity-Based Purification
Chemical and affinity-based methods leverage molecular characteristics rather than bulk properties, providing alternatives to physical separation. Polymer-based precipitation relies on adding hydrophilic polymers, such as polyethylene glycol (PEG), to the solution. These polymers selectively exclude water, thereby reducing the solubility of the vesicles in the resulting solution.
The concentrated PEG creates a crowded environment, encouraging exosomes to aggregate and precipitate out of the solution, which is then collected by low-speed centrifugation. This method is high-throughput and relatively simple, making it suitable for processing large numbers of samples. However, the resulting pellet may contain co-precipitated proteins and polymer residues that can interfere with subsequent molecular analysis.
Immunoaffinity capture leverages specific molecular markers displayed on the exosome surface for highly selective isolation. This technique uses antibodies immobilized on magnetic beads or columns that recognize common exosomal surface proteins. Researchers often target tetraspanin proteins like CD9, CD63, or CD81, which are highly abundant on the exosome membrane. Target exosomes bind to the antibody-coated surface, and contaminants are washed away, resulting in highly specific isolation. While immunoaffinity yields high purity, the binding interaction can potentially alter the biological function of the isolated vesicles. Furthermore, the yield is often lower compared to physical methods because only a subset of exosomes expressing the specific marker is captured.
Analyzing the Purity of Isolated Exosomes
After isolation, quality control methods confirm the successful purification and integrity of the vesicles by characterizing physical properties and verifying molecular content. Nanoparticle Tracking Analysis (NTA) determines the concentration and size distribution of particles in the solution. NTA confirms that most isolated vesicles fall within the expected 30–150 nm range, providing quantitative data on yield and homogeneity. Transmission Electron Microscopy (TEM) provides high-resolution images, allowing researchers to visually verify the characteristic cup-shaped or spherical morphology and structural integrity of the isolated exosomes.
Molecular characterization typically involves Western blotting to verify specific exosome-associated proteins. Researchers look for positive markers like the cytosolic protein TSG101 or the surface tetraspanins CD9, CD63, and CD81 to confirm identity. Equally important is confirming the absence of common cellular contaminants that would indicate a failed purification. For example, the presence of the endoplasmic reticulum marker Calnexin or the nuclear protein Histone H3 indicates significant contamination from cellular debris, suggesting a need to refine the initial purification steps.