Recent Advances in Exosome Characterization and Measurement Technologies
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 from 40 to 100 nm in diameter and have a density between 1.10 and 1.18 g/ml. They carry various proteins, mRNAs, and miRNAs and play 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, concentration, and fluorescent properties.
1. Exosome Isolation and Method Evaluation
Exosome isolation remains challenging due to the need to balance yield, purity, and biological activity. Common methods include centrifugation, filtration, density gradient centrifugation, immunomagnetic separation, and chromatography, each with its own strengths and limitations.
1.1 Centrifugation
Centrifugation is the most widely used method. It involves sequential centrifugation steps at increasing speeds to remove debris and larger particles before isolating exosomes at 100,000 g. While this method yields large quantities, it often results in low purity, as exosomes may aggregate or be confused with microbubbles. Some studies have shown that not all isolated vesicles are true exosomes.
1.2 Filtration and Centrifugation
This technique uses ultrafiltration membranes with varying molecular weight cut-offs (MWCO) to separate exosomes based on size. It is simple, fast, and preserves exosome integrity, but still faces challenges in achieving high purity.
1.3 Density Gradient Centrifugation
Using sucrose gradients, this method separates exosomes by their density. It produces highly pure exosomes, though the process is time-consuming and requires careful preparation. The yield is also relatively low.
1.4 Immunomagnetic Beads
Immunomagnetic beads coated with antibodies against exosomal markers like CD9, CD63, and Alix can specifically capture exosomes. This method is efficient and selective, but the use of non-physiological conditions may affect exosome function.
1.5 Chromatography
Chromatographic techniques separate exosomes based on size, offering high uniformity. However, they require specialized equipment and are not widely adopted in routine research.
2. Comparison of Exosome Measurement Techniques
Accurate characterization of exosomes is essential for understanding their role in disease and therapy. Several techniques are available, each with distinct capabilities.
2.1 Electron Microscopy (EM)
Scanning electron microscopy (SEM) provides high-resolution images of exosome morphology. However, sample preparation is complex and time-consuming, making it unsuitable for high-throughput analysis. Additionally, it cannot measure exosome concentration directly.
2.2 Dynamic Light Scattering (DLS)
DLS measures particle size by analyzing light intensity fluctuations caused by Brownian motion. It is fast and easy to use, but it struggles with polydisperse samples, where larger particles can mask smaller ones. It also lacks the ability to quantify exosome concentration accurately.
2.3 Nanoparticle Tracking Analysis (NTA)
NTA is a powerful and emerging technique that tracks individual nanoparticles in real-time. It provides both size distribution and concentration data. NTA works by illuminating a sample with a laser and capturing the Brownian motion of particles through a microscope. Software then calculates hydrodynamic radius and concentration using the Stokes-Einstein equation.
NTA has a lower detection limit of about 30–40 nm, suitable for exosomes. It can also distinguish between different particle sizes in a mixture, as demonstrated by experiments with polystyrene beads of 100 nm and 300 nm. Furthermore, NTA supports fluorescence detection, enabling the identification of labeled exosomes in complex environments such as serum. Compared to flow cytometry, NTA offers higher resolution and better sensitivity for small fluorescent particles.
3. Conclusion
Exosome research is still in its early stages, but its potential in diagnostics and therapeutics is promising. To fully realize this potential, accurate and reliable measurement tools are essential. While traditional methods have their place, NTA stands out for its high resolution, real-time analysis, and ability to measure both size and concentration. Its unique capabilities make it an ideal tool for advancing exosome research and improving clinical applications.
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