Table 1.

EV isolation methods.

Method	Brief Technique Description	Advantages	Disadvantages	Ref.
UC	The sample is centrifuged (300× g for 10 min; 2000× g for 20 min to eliminate dead cells; and 10,000× g for 30 min to remove debris). EVs are isolated by UC at 100,000× g for 60 min at 4 °C. The pellet is resuspended in PBS and re-centrifuged at 100,000× g for 60 min	It represents the most widely used technique for EV isolation in basic and translational research. For these reasons it is highly standardized and reproducible	Generation of EV aggregates, contamination of the EV sample with smaller size particles. The whole isolation process is time consuming	[16] [17] [18] [19]
DG	A density gradient is generated by adding decreasing concentration of iodixanol or sucrose in a tube. The sample is then added on the top of the gradient and is centrifuged at 100,000× g for 18 h at 4 °C. After centrifugation, the recovered gradient fractions are diluted in PBS. An additional 3 h centrifugation is performed at 100,000× g	It represents a highly efficient method allowing to isolate purified EVs	It is more time consuming and more labour- intensive than standard UC. This consideration can make its introduction in the clinical practice difficult	[16] [17]
SEC	SEC separates molecules based on their size by filtration through a resin-packed column. After sample centrifugation (1100× g for 10 min) and filtration, samples are loaded in size exclusion chromatography columns	SEC isolates high purity, functionally and structurally preserved EVs. It has been defined as one of the best methods for EV isolation	Compared to other methods, the total EV yield is lower and in the clinical setting, higher volumes of biological fluid may be needed to overcome this issue	[18,20]
Polymer-based precipitation (e.g.,: Exoquick)	The method is based on the use of PEG to capture EVs. After PEG preparation, the sample undergoes isolation using 10% PEG solution at 4 °C for 2 h. After incubation, samples are centrifuged at low-speed × 10 min. To ensure major purity a double-step approach can be followed	It represents a fast, easy, and widespread method for EV isolation. It also easily allows a simultaneous isolation from multiple samples	EV obtained with this method have higher chances of being contaminated and therefore less suitable for omics-based analysis	[14,20,21]
Immunoaffinity capture	The immunoaffinity capture method relies on the isolation of EVs based on the expression of surface markers. It commonly uses antibodies against specific EV surface proteins, e.g., tetraspanin: CD9, CD63, and CD81	The immunoaffinity capture isolation allows EV-specific isolation based on their surface markers. It can also be used as an additional step after UC to enhance EV purity	The removal of antibodies from EV surface could damage EVs. The selection of specific EVs could eventually not reflect the characteristics of all separated EVs. It is more expensive compared to other methods.	[17,20]
Microfluidic isolation	It consists in a high-throughput method using microfluidic devices to isolate EVs based on several principles: immunoaffinity, size, and density. The most used is the immuno-microfluidic technique, which is similar to the immune-affinity-capture isolation method. Antibodies immobilized on microfluidic devices serve to EV isolation	Relatively fast and high purity EV isolation can be obtained with this technique. It is a new and promising method for EV isolation	Shares with immunoaffinity the same disadvantages. It is expensive and standardization is still lacking	[18]

UC: Ultracentrifugation, DG: Density Gradient Centrifugation, SEC: Size Exclusion Chromatography.