Table 1.

Summary of EV separation methods and examples of their utilization in in vivo drug delivery studies whenever available.

Method	Principle	Advantages	Disadvantages	In vivo drug delivery examples
Tangential flow filtration	Size	Highly scalable, automatable, gentle, choice of membrane, disposable devices	Unable to remove EV-sized impurities, non-specific interactions with membranes	[23]
Ultrafiltration	Size	Rapid, scalable, choice of membrane, no dedicated major equipment, disposable devices	Clogging, sample loss, EV damage, unable to remove EV-sized impurities	UF, UC, sucrose cushion [103]
Size-exclusion chromatography	Size	Gentle, scalable, good separation, removes soluble proteins and small molecules	Sample dilution might require post-separation concentration, low yield, limited sample capacity, unable to remove EV-sized impurities	NA
Bind-elute chromatography	Size/affinity	Rapid, scalable, gentle, one-step elution, removes soluble proteins and small molecules	Low yield, limited sample capacity	NA
Ion-exchange chromatography	Charge	Rapid, scalable, one-step elution	Low specificity for EVs, separation conditions need to be optimized, might require post-separation buffer exchange and concentration	NA
Affinity chromatography	Affinity	Rapid, scalable	Unclear purity, elution might damage vesicles	[104]
(Immuno)affinity	Affinity	Rapid, high purity, no specialized equipment, specific capture of engineered EVs	Costly, low throughput, low yield, unclear scalability, a priori knowledge of surface markers is necessary, affinity reagents need to be removed without damaging EVs	NA
Differential ultracentrifugation	Sedimentation	Inexpensive, easy to use	Low throughput, low scalability, needs specialized equipment, unable to remove EV-sized impurities, potential EV damage and aggregation	[105-107]
Density gradient centrifugation	Density	Commonly used method, inexpensive, high purity, often used in combination with other methods	Low throughput, low scalability, needs specialized equipment, low yield, lengthy and cumbersome procedure	Density gradient [108] Density cushion [109, 110]
Precipitation	Solubility	Highly scalable, rapid, no specialized equipment, easy to use, inexpensive	Low purity, co-separates soluble non-EV material, precipitation reagent needs to be removed	[111-113]