Methods of Exosome Isolation

As major biomarkers in the early diagnosis of cancer, as well as inflammatory, cardiovascular, and neurodegenerative diseases, exosomes are a prominent area of disease research. But in order for this research to occur, an efficient and reliable method of exosome isolation is required. However, despite this critical need, the heterogeneity of exosomes, the complexity of biological fluids, and the presence of nanoscale contaminants mean that exosome separation is no mean feat. Nevertheless, various methods of exosome isolation exist, and the methods are continually improving. The key is choosing the method that best suits your needs.

”Ultracentrifugation is the classic approach used for isolating exosomes,” explains John W. Ludlow, Vice President Regenerative Medicine at Zen-Bio, and “many still consider this method the gold standard.” Other methods such as density gradient centrifugation, ultrafiltration, size-exclusion chromatography, immunoaffinity, and precipitation also lie at the center of traditional exosome isolation. But despite their widespread use and wide sample compatibility, these traditional methods are often time-consuming and labor intensive, require specialized instrumentation, and can result in low yields. Furthermore, exosomes can be damaged by centrifugal force, compromising their structure and function, and limiting downstream analysis.

In recent years, advances in microfluidics combined with technologies such as nanolithography, immunomagnetic beads, covalent chemistry, DNAzyme probes, and negative magnetic electrophoresis, have led to significant improvements in exosome isolation. Emerging isolation techniques offer benefits of higher yield and purity, and increased speed and simplicity, while maintaining the structural integrity and function of exosomes. These techniques can be expensive, however, with limited sample capacity, and exosomes can be co-isolated with other particles.

“When selecting a technique for exosome isolation, the main thing to consider is the application that you are using it for,” explains Afrida Rahman-Enyart, Scientific Liaison and Product Manager at Proteintech Group. “For example, for omics research, density gradient centrifugation and size-exclusion chromatography are superior, whereas polymer-based precipitation would not be ideal for this scenario since purity is quite low.” Alternatively, if your research looks at specific subclasses of exosomes, then immunoaffinity would be the obvious choice, however the cost and time-consuming nature of the technique limits its benefit if subclasses are not at the heart of your research.

”Considering the sample source, desired purity, and downstream applications,” as well as “assessing the scalability, cost-effectiveness, and compatibility with downstream analyses” are also key, according to Agnė Vaitkevičienė, Co-Founder and CEO, and Karolina Karl, Business Development Director at Memel Biotech. Immunomagnetic separation and covalent chemistry techniques, for example, are easy to combine with analysis tools, but their limited sample capacity limit scalability.

Furthermore, you should also consider “the combination of several techniques,” Rahman-Enyart says, as “Optimal yield and purity are difficult to achieve by a single method,”  Ludlow explains. Coupling centrifugation with size-exclusion chromatography for example, can help reduce the impurities from small contaminants and result in highly purified exosomes.

”Exosomes and their applications are a dynamic and growing area of research that have the potential to disrupt multiple areas of medicine,” but as Vaitkevičienė explains “There is no escaping the fact that they are complex and challenging to manufacture and deliver.” Memel Biotech, in collaboration with partner companies specializing in exosome research, are looking for ways to enhance the isolation of exosomes. Launched this year, their state-of-art facility in Lithuania aims to manufacture high-quality and GMP-compliant exosomes to support the creation of optimal therapeutic and diagnostic solutions. It would seem that further developments in the methods of exosome isolation are still to come.

Different Methods of Exosome Isolation