Recent Advances in Exosome Characterization and Measurement Technology
Author: Zhang Shuai, Bioscience Expert at Malvern Instruments, UK
Abstract: Exosomes were first identified in the supernatant of cultured sheep red blood cells. These small vesicles typically range in size from 40 to 100 nm with a density between 1.10 and 1.18 g/ml. They carry various biomolecules such as proteins, mRNAs, and miRNAs, playing key roles in cell communication, migration, angiogenesis, and tumor progression. As potential natural drug carriers, exosomes hold great promise for clinical applications. However, limitations in current measurement techniques have hindered their full exploration. This article reviews common exosome isolation methods, compares different characterization techniques, and highlights the advantages of nanoparticle tracking analysis (NTA) in studying exosome size and concentration.
1. Exosome Isolation and Method Evaluation
Currently, no single method can simultaneously ensure high yield, purity, and biological activity of exosomes. Common approaches include centrifugation, filtration, density gradient centrifugation, immunomagnetic beads, and chromatography. Each has its own strengths and limitations.
1.1 Centrifugation
This is the most widely used technique. It involves sequential centrifugation at increasing speeds (300g, 2000g, 10,000g, and finally 100,000g) to pellet exosomes. While this method yields large quantities, it often results in low purity due to contamination by microbubbles and other particles. Some studies suggest that not all collected vesicles are true exosomes.
1.2 Filtration and Centrifugation
This approach uses ultrafiltration membranes with different molecular weight cut-offs (MWCO) to separate exosomes from larger molecules. It is simple, time-efficient, and preserves exosome integrity. However, it still faces challenges with purity and potential cross-contamination.
1.3 Density Gradient Centrifugation
This method separates exosomes based on their density using gradients like sucrose. After ultracentrifugation, exosomes collect at their iso-density zone (1.10–1.18 g/ml). This technique provides high-purity exosomes but is labor-intensive, time-consuming, and yields smaller amounts.
1.4 Immunomagnetic Beads
Exosomes are captured using magnetic beads coated with specific antibodies (e.g., CD9, CD63, Alix). This method is selective, easy to perform, and does not require expensive equipment. However, non-physiological conditions during the process may affect exosome function and complicate downstream experiments.
1.5 Chromatography
Chromatographic techniques separate exosomes based on size. While they produce uniform-sized particles suitable for electron microscopy, they require specialized equipment and are less commonly used in routine research.
2. Comparison of Exosome Measurement Techniques
2.1 Scanning Electron Microscopy (SEM)
SEM provides high-resolution images of exosome morphology. However, sample preparation is complex and time-consuming, making it unsuitable for rapid or high-throughput analysis. It also cannot accurately measure exosome concentration.
2.2 Dynamic Light Scattering (DLS)
DLS measures particle size based on light intensity fluctuations caused by Brownian motion. It is fast and simple, but struggles with polydisperse samples. It cannot distinguish between different sizes in complex mixtures and lacks concentration measurement capabilities.
2.3 Nanoparticle Tracking Analysis (NTA)
NTA is a powerful new technique that tracks individual nanoparticles in real-time. Using laser illumination and video imaging, it calculates particle size and concentration based on Brownian motion. NTA offers high resolution, accurate concentration measurements, and the ability to detect fluorescent particles.
NTA works by illuminating a sample with a laser beam and capturing the movement of particles through a microscope camera. The software tracks each particle, calculates its hydrodynamic radius using the Stokes-Einstein equation, and determines concentration. It can distinguish between particles of different sizes and even identify fluorescently labeled exosomes in complex backgrounds like serum.
For example, when measuring a mixture of 100 nm and 300 nm polystyrene beads, NTA clearly separated the two populations. It also showed good correlation between measured and actual concentrations in monodisperse samples. Additionally, NTA’s fluorescence mode allows for detection of exosomes labeled with fluorescent antibodies, offering better resolution than flow cytometry.
3. Conclusion
Although exosome research is still in its early stages, it holds significant potential for clinical diagnostics and therapeutics. Accurate and efficient measurement of exosome size and concentration remains a challenge. NTA offers a promising solution, providing real-time, high-resolution, and reliable data. Its ability to analyze complex samples and detect fluorescent markers makes it a valuable tool for advancing exosome research.
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