TABLE 1.

Methods of EV isolation, preparation and purification and their pitfalls.

Method	Strengths	Pitfalls	References
Sequential centrifugation with fixed angle rotors	• easy to perform with standard equipment	• shear forces can damage EVs, lead to leakiness and loss of cargo, and generation of inside-out vesicles (thereby false-positive exposure of phosphatidylserine)	(Cvjetkovic et al., 2014; Linares et al., 2015; Livshits et al., 2015; Szatanek et al., 2015; Théry et al., 2018; Lee et al., 2019)
	• fairly fast	• ultracentrifugation as final enrichment can lead to aggregation of EVs
	• enrichment can be achieved by final ultracentrifugation
Sequential centrifugation with swing-out rotors	• easy to perform with standard equipment	• centrifugation without a high-density bottom layer can still lead to EV damage	(Szatanek et al., 2015; Li et al., 2018; Théry et al., 2018; Lee et al., 2019)
	• fairly fast	• A sucrose solution as bottom cushion has strong osmotic strength, which might affect EVs
	• enrichment can be achieved by final ultracentrifugation
Float-up gradient centrifugation	• still easy to perform with standard equipment	• The gradient material might have unknown influences on EV functions, thus inert gradient substances should be used	(Iwai et al., 2016; Li et al., 2018; Théry et al., 2018; Lee et al., 2019; Zhang et al., 2020)
	• enrichment at the interface of two layers with different densities is possible
	• Purification effect (from associated proteins or aggregates) by the float-up process
Precipitation with polymers such as PEG	• Fast method	• Precipitation alone results in low purity and contamination with lipoprotein particles and should therefore be combined with other methods	(Szatanek et al., 2015; Théry et al., 2018; Zhang et al., 2020)
	• Kits are available
Size-exclusion chromatography	• mild purification with hardly any damage of EVs	• Rather time-consuming and equipment not lab standard	(Nordin et al., 2015; Szatanek et al., 2015; Théry et al., 2018; Lee et al., 2019; Zhang et al., 2020)