AAT Bioquest

What are the techniques used in the isolation of exosomes?

Posted July 21, 2023


Several different techniques can be used in the isolation of exosomes. Each of these has its own pros and cons. 


This technique involves spinning down the biological fluid containing exosomes at high speeds in a centrifuge. The process separates exosomes from other particles based on their size and density. Although it is currently a gold standard for exosome isolation, it yields only moderate purity because of the overlap in density ranges. While increasing centrifugal speed can improve the purity, it increases the risk of damaging the exosomes. 


Ultrafiltration relies on the size exclusion principle to isolate exosomes based on their size. In this technique, pressure is applied to drive the biological fluid through membranes with extremely small pores measuring approximately100 nm. The membrane holds the larger particles back while allowing the smaller-sized exosomes to pass through. Although this technique is faster than ultracentrifugation, the applied pressure combined with potential membrane adhesion can result in damage and loss of exosomes. 

Exosome ultrafiltration methods include centrifugal, sequential, tangential flow, and tandem filtration. 


Exosome precipitation techniques involve the use of an aqueous PEG (Poly-Ethylene Glycol) solution to facilitate exosome aggregation and subsequent precipitation using low-speed centrifugation. Although this technique results in relatively high yield, purity and specificity is largely lost because of co-precipitation of soluble non-exosomal proteins. To overcome the drawbacks associated with this technique, PEG-based isolation is often combined with other exosome-isolation techniques. 

Immunoaffinity Capture

Immunoaffinity-based methods use specific antibodies that recognize and bind to unique exosomal surface markers. Multiwell plates, magnetic beads, or other solid supports coated with these antibodies are added to the sample, allowing the exosomes to bind to the antibodies. The bound exosomes can then be isolated using a magnetic field or other separation methods. This technique offers high specificity and allows for the isolation of exosomes based on their specific surface markers.


This technique isolates exosomes using microfluidic devices that use tiny channels and precise fluid control to separate exosomes. The isolation is based on multiple principles including size, density, and immunoaffinity. These devices can achieve high-resolution separation and offer the advantage of requiring smaller sample volumes. However, the need for specialized, expensive equipment makes this a less accessible option.  

Additional resources

Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis

DiOC16(3) perchlorate [3,3-Dihexadecyloxacarbocyanine perchlorate]

DiR iodide [1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide]