Isolation of exosomes is a fundamental step in the investigation of cellular biogenesis, biomarkers and potential therapeutic applications. There are several methods to carry out this task, but two of the most widely used are ultracentrifugation and immunoaffinity. In this article, we discuss their main differences, advantages and limitations.
Ultracentrifugation: the traditional method
Differential ultracentrifugation has been the reference method for exosome isolation due to its ability to separate vesicles according to their size and density. It is based on the application of high centrifugation speeds (100,000 g or more) to sediment the exosomes.
Advantages of ultracentrifugation
-Researchers can obtain large volumes of exosomes.
-Does not require expensive reagents
-It is a standardised method in many laboratories
Limitations of ultracentrifugation
-Can lead to contamination with other extracellular vesicles and soluble proteins
-It is a laborious process and requires specialised equipment
May affect the structural integrity of exosomes due to high g-force
-Variability in exosome recovery and purity can affect reproducibility of studies
Immunoaffinity: specific targeting of exosomes
Immunoaffinity isolation is based on the interaction between antibodies and specific surface proteins of exosomes. It uses magnetic beads coated with antibodies directed against exosome markers, such as CD9, CD63 and CD81.
Advantages of immunoaffinity
-High specificity in the selection of exosomes
-Reduces contamination with other vesicles and proteins
-Allows for rapid and reproducible isolation
-Ideal for biomarker studies, as highly purified exosomes can be obtained.
Limitations of immunoaffinity
-Not ideal for large samples, as the amount of exosomes recovered is limited.
-Can be costly due to the use of specialised antibodies and reatcives
-Dependence on the expression of the selected biomarkers
-Loss of exosomes that do not express the selected surface markers may occur, which could bias the results.
Other exosome isolation techniques
In addition to ultracentrifugation and immunoaffinity, there are other methods of exosome isolation that can be used depending on the research context:
-Polymer precipitation: uses polymeric reagents to induce exosome aggregation, facilitating their collection by low-speed centrifugation.
-Size filtration: This is based on the use of membranes with specific pores to retain exosomes while removing other larger or smaller particles.
-Size exclusion chromatography (SEC): A gentle method that allows the separation of exosomes without damaging their integrity, preserving their biological functionality.
Conclusion: Which method to choose?
The choice between ultracentrigugation and immunoaffinity depends on the objective of the study. If a large amount of exosomes is needed and removal of contaminants is not a priority, ultracentrifugation remains a viable option. On the other hand, if high purity and specific selection are required, immunoaffinity is the best alternative.
To ensure quality research, it is recommended to evaluate the characteristics of each method and, in some cases, to combine techniques to improve isolation efficiency and purity.
At Immunostep, we develop innovative solutions for exosome isolation with high specificity, allowing researchers to optimise their protocols and obtain reliable results. Find out more about our technologies on our website and optimise your exosome research.