Exosomes are nanosized, membrane-bound sacs released by nearly all cell types, typically measuring between 30 and 150 nanometers in diameter. These structures originate within the cell’s endosomal compartment and are extruded into the surrounding biological fluid through exocytosis. Exosomes function as messengers, carrying a cargo of proteins, lipids, messenger RNA (mRNA), and microRNA (miRNA) from their parent cell. This cargo is delivered to recipient cells, influencing their function in both healthy processes and disease states. Researchers focus on harvesting exosomes because the molecules they carry offer a snapshot of the originating cell’s condition, presenting a platform for non-invasive disease diagnosis and targeted drug delivery. Their stability in bodily fluids makes them attractive biomarkers for conditions like cancer, neurodegenerative disorders, and cardiovascular disease.
Preparing Biological Samples for Isolation
Before harvesting, biological material must undergo preparation to remove large, contaminating particles. Exosomes are found in numerous biological fluids, including blood plasma, serum, urine, saliva, and cell culture media. The initial step involves sequential low-speed centrifugation, which exploits size and density differences between cellular components.
The first spin, often at 300 x g, pellets intact cells and large cellular debris. The supernatant is then subjected to subsequent spins at incrementally higher forces, such as 2,000 x g and 10,000 x g. These steps remove remaining large particles, such as dead cells and apoptotic bodies, which are larger than exosomes. This prevents these structures from co-isolating with the exosomes later.
Filtration is an alternative or complementary approach using membranes with defined pore sizes to physically exclude larger particles. Passing the sample through a 0.22 micrometer (µm) syringe filter removes residual bacteria and fine debris that survived centrifugation. Ensuring the starting material is free of these contaminants enhances the purity of the final exosome preparation, regardless of the downstream harvesting method chosen.
The Standard Approach Ultracentrifugation
Ultracentrifugation is the most established technique for harvesting exosomes and is considered the gold standard for high-purity research. This approach uses extremely high centrifugal forces to separate components based on their sedimentation rate, which depends on size, shape, and density. Differential ultracentrifugation (DUC) is the standard workflow, involving a series of spins at progressively higher speeds.
After pre-cleaning, the supernatant is subjected to forces exceeding 100,000 x g for 90 minutes or more. This causes the small, low-density exosomes to pellet at the bottom of the tube. The pellet is washed, resuspended in a buffer, and the high-speed spin is often repeated to increase purity. While DUC is effective, it often co-isolates non-exosomal particles like protein aggregates and lipoproteins, which share a similar size and sedimentation rate.
To achieve higher resolution, researchers employ density gradient ultracentrifugation (DGUC). This technique separates exosomes by their buoyant density, typically between 1.12 and 1.18 grams per milliliter (g/mL). The sample is layered onto a gradient medium, often sucrose or iodixanol, creating layers of increasing density. Exosomes migrate until they reach the point where their density matches the surrounding medium. Collecting the fraction at this specific density yields a highly purified population, distinguishing them from denser protein complexes.
Alternative and Specialized Harvesting Methods
Ultracentrifugation has limitations, including the need for specialized equipment, long processing times, and potential damage to exosome structure. Several alternative methods address these issues.
Polymeric Precipitation
Polymeric precipitation is an accessible method involving the addition of a hydrophilic polymer, such as polyethylene glycol (PEG), to the sample. The polymer reduces the solubility of the exosomes, causing them to aggregate and precipitate out of the solution. These aggregates are collected using low-speed centrifugation, bypassing the need for a high-speed ultracentrifuge and allowing for high-throughput processing.
Size Exclusion Chromatography (SEC)
SEC separates particles by their hydrodynamic radius as they pass through a packed column. The sample is loaded onto the column, and smaller components, including exosomes, are temporarily retained within the pores of the column resin. Larger components pass through more quickly. This gentle technique avoids high mechanical stress and results in preparations with high integrity and purity, though it yields a more dilute final product.
Immunoaffinity Capture
Immunoaffinity capture is a highly specific isolation technique leveraging unique protein markers displayed on the exosome surface. This method uses antibodies that recognize common exosomal proteins, such as the tetraspanins CD9, CD63, or CD81. These antibodies are bound to a solid support, like magnetic beads or a microfluidic chip. When the sample flows over the surface, only exosomes expressing the target marker bind, allowing for the selective isolation of specific subpopulations.
Confirming Successful Exosome Isolation
Researchers must confirm that the isolated material is exosomes and not residual debris or protein aggregates. This validation relies on techniques that characterize the physical and biochemical properties of the collected particles.
Size and concentration analysis uses Nanoparticle Tracking Analysis (NTA). This instrument uses light scattering to visualize and track the movement of individual particles, determining their size distribution and total concentration. Successful isolation shows a particle size profile predominantly within the expected 30 to 150 nanometer range.
Biochemical characterization is accomplished using Western Blotting, which detects established exosomal signature proteins. Researchers look for the enrichment of positive markers:
- The cytosolic protein TSG101
- The membrane protein Alix
- The tetraspanin proteins CD9
- The tetraspanin proteins CD63
The absence of markers associated with other cellular compartments, such as the endoplasmic reticulum protein Calnexin, confirms the preparation is free of contamination from broken cells. Finally, electron microscopy, including Transmission Electron Microscopy (TEM), provides visual confirmation of the characteristic morphology. TEM images reveal the expected spherical or cup-shaped structure of the harvested exosomes, providing physical validation.