Cells in the body constantly communicate by sending and receiving molecular messages, with exosomes acting as microscopic mail carriers in this network. These tiny vesicles, released by nearly all cell types, are enclosed in a lipid bilayer. They carry a cargo of proteins, lipids, and genetic material from their parent cell, delivering this payload to influence a recipient cell’s function.
The specific contents of an exosome can reflect the health of its cell of origin, offering a window into disease processes. This makes them promising candidates for diagnostic tools, where a blood sample could reveal disease markers. Scientists are also exploring their use in therapeutics by loading exosomes with specific drugs for direct delivery to target cells. To realize this potential, researchers must first efficiently and purely collect them.
Sources for Exosome Harvesting
The choice of source for exosome isolation is dictated by the research question or intended application. A common source is cell culture media, the nutrient-rich liquid used to grow cells in a laboratory. Harvesting from cell cultures provides a controlled environment, allowing scientists to collect exosomes from a specific cell type, such as mesenchymal stem cells, which produce them in large quantities.
For disease diagnostics, researchers use accessible biofluids. Blood is a common choice, with plasma and serum serving as rich sources of exosomes released from cells throughout the body, making it valuable for identifying systemic disease biomarkers. Urine is useful for studying conditions of the kidneys and urinary tract, as it contains exosomes from cells lining these systems. Saliva is another non-invasive source providing exosomes from the oral cavity.
The Standard Harvesting Method: Ultracentrifugation
The benchmark for isolating exosomes is differential ultracentrifugation, a multi-step process using centrifugal force to separate particles based on size and density. It is considered the gold standard because it yields a relatively pure exosome population, though it requires specialized equipment and is a lengthy procedure. The process is carried out at cold temperatures (around 4°C) to preserve the integrity of the exosomes and their cargo.
The process begins with the biological sample, such as cell culture media or blood plasma. The first step involves low-speed spins, around 300 to 2,000 times the force of gravity (x g), to remove the largest components like whole cells and large debris. After this pellet is discarded, the remaining liquid, or supernatant, is subjected to a more powerful centrifugation step at 10,000 to 20,000 x g. This higher speed pellets smaller debris and larger vesicles, which are also discarded, leaving a supernatant enriched with exosome-sized particles.
The final step is ultracentrifugation, where the supernatant is spun at extremely high speeds, often exceeding 100,000 x g for an hour or more. This force compels the tiny exosomes to migrate to the bottom of the tube, forming a small pellet. After this high-speed spin, the supernatant is removed, leaving behind the concentrated exosomes. This pellet is then washed with a buffer solution and spun again at high speed to remove any remaining contaminating proteins, resulting in a purified exosome sample.
Alternative Harvesting Techniques
Other methods have been developed to isolate exosomes, offering advantages in speed, cost, or specificity. One technique is size-exclusion chromatography (SEC), which functions like a molecular sieve. The sample is passed through a column packed with porous beads. Larger particles, including exosomes, cannot enter the small pores and travel quickly through the column to be collected first. Smaller molecules, like free proteins, enter the pores, slowing their journey and separating them from the exosome fraction.
Another approach involves precipitation, where a polymer like polyethylene glycol (PEG) is added to the sample. PEG works by binding water molecules, which reduces the solubility of the exosomes and causes them to precipitate. These precipitated exosomes can then be collected by a low-speed centrifugation step. This technique is faster and does not require an ultracentrifuge but carries a higher risk of co-precipitating other proteins.
A more targeted method is immunoaffinity capture, which isolates exosomes based on specific proteins on their surface. This technique uses microscopic magnetic beads coated with antibodies designed to bind to common exosome surface markers, like CD9, CD63, or CD81. When mixed with a sample, the beads selectively latch onto the exosomes. A magnetic field is then applied to hold the beads and attached exosomes in place while contaminants are washed away, yielding a highly specific sub-population.
Verifying the Harvest
Once a sample of exosomes has been isolated, it is necessary to confirm the harvest was successful and the final product is pure. This quality control step involves several analytical techniques to characterize the collected particles. This ensures that downstream experiments are conducted with the correct material.
One primary verification method is direct visualization using electron microscopes. Transmission electron microscopy allows scientists to see the particles, confirming they have the expected small size, between 30 and 150 nanometers. It also confirms the characteristic cup-shaped morphology that results from the sample preparation process, providing visual evidence of exosome-like structures.
Researchers must also measure the size distribution and concentration of the particles. A common technique for this is Nanoparticle Tracking Analysis (NTA), where a laser illuminates the particles in a liquid, and a camera records their movement. The instrument’s software tracks the motion of each particle to calculate its size and determines the overall particle concentration, confirming the vesicles fall within the expected size range.
Finally, the molecular content of the harvested particles is analyzed to confirm their identity. This is done using Western blotting, which detects the presence of specific proteins. Scientists test the sample for proteins known to be enriched within exosomes, such as CD9 and CD63. They also test for the absence of proteins from other parts of the cell to ensure the sample is not contaminated with cellular debris.