Table 4.

Summary of the main studies on exosomes using FFF. The table highlights the experimental set ups, including FFF platforms and additional offline techniques.

Exosome Matrix	FFF Platform	Additional Techniques (Offline)	Results
Exosomes form human neural stem cells.	mFI-AFlFFF-UV (miniaturized frit inlet asymmetrical FlFFF UV-coupled)	TEM; LC-ESI-MS-MS	Exosome subpopulations larger than ∼50 nm were morphologically distinct from those smaller than ∼50 nm. Each exosome fraction showed a different protein pattern.	[179]
Non-labeled B16-F10 exosomes from an aggressive mouse melanoma cell culture line.	AF4-UV-MALS	DLS; TEM	Label-free separation of exosomes into subfractions and corresponding size characterization.	[171]
Lyophilized exosome standard HBM-BLCL21-30 [55] purified from the culture supernatant of an EBV-transformed. lymphoblastoid B cell line (HansaBioMed, Tallinn, Estonia).	AF4-UV-MALS	DLS; NTA (nanoparticle tracking analysis); TEM	Significant influence of crossflow conditions and channel thickness on fractionation quality. Identification, separation, and size-characterization of two exosomes subpopulations.	[55]
Exosomes isolated form the human urine of Pca patients and healthy controls.	AF4-UV	TEM; Western Blotting; UPLC-ESI-MS/MS	Exosome separation and size distribution characterization. The Lipidomic analysis of selected fractions indicated differences in lipidic content and composition between the exosomes of patients and health controls.	[167]
B16-F10 melanoma-derived exosomes.	AF4-QUELS-UV	NTA; TEM; Mobius Zetasizer AFM; Blotting and MS Techniques; Odyssey Imaging System	The separation of two discernible exosome subpopulations, Exo-S and Exo-L, and the identification of distinct exomeres, which differ in size and content from other reported particles. Proteins, glycans, lipids, and nucleic acids are selectively packaged in exomeres.	[178]
Purified human A375 melanoma exosomes.	Cy-El-FFF-UV-MALS	Mobius Zetasizer	The effect of buffer solution composition and dilution on exosome properties and separation.	[180]
Extracellular vesicles from human plasma.	AF4-UV-MALS	Western Blotting; TEM; HPLC-C18; LC-ESI-MS/MS	Human plasma contains more EVs than the paired serum and shows age- and gender-independent individual variability of the amount of EVs in human plasma. Most of the proteins identified in the EVs from human plasma were involved in extracellular matrix structural constituents and associated with the ECM–receptor interaction pathway.	[181]
Exosomes isolated from human serum samples.	AF4-UV-MALS	DLS; Western Blotting; nUHPLC-ESI-MS/M	The evaluation of the ability of ultrafiltration and ultracentrifugation in exosome isolation from serum. A simple centrifugation followed by UF offered advantages, such as faster preparation and higher exosomal recovery, with smaller sample volumes than the UC method. However, the removal of lipoproteins seemed more efficient with UC than UF.	[177]
Samples obtained from the ultracentrifugation of the culture medium of murine myoblasts (C2C12).	HF5-UV-FLD-MALS	NTA; TEM; Western Blotting	The overall characterization of small and large EVs in all the fractions obtained through ultracentrifugation. The identification of an otherwise-hidden rod-shaped species carrying nucleic content, was found predominantly in the densest SEV fractions, which could potentially correspond to exomeres.	[40]
Fractions of exosomes and microvesicles were isolated from the culture media of DU145 cells using a series of centrifugation methods, including UC.	AF4-UV-MALS	nUHPLC-ESI-MS/MS; TEM; Western Blotting	Both UC and UF methods can be utilized for the initial isolation of EVs from cell culture media prior to the FlFFF separation of exosomes and microvesicles; however, UF was found to be more efficient than UC. The hyphenation of FlFFF with ESI-MS/MS allowed for the selective detection of lipid targets and specific biomarkers.	[182]