Isolation of Extracellular Vesicles: General Methodologies and Latest Trends

Table 3

Characterization and comparison of different methods for EV isolation according to EV size and amount, representation of EV markers, and data on the proteins, RNA, and microRNA contained in isolated EV preparations.

	EV size	EV quantity	EV markers	Protein	RNA	miRNA
(1.1) Ultracentrifugation (UС)	20–100 nm; on the average, 53 nm (cell medium) [51]; 40–100 nm + larger EVs (cell medium) [36]; 50–250 nm (cell medium) [57]; <300 nm; mainly, 30–150 nm (cell medium and plasma) [89]; and 90.1 ± 10.0 nm (cell medium) [90]; 70–110 nm (plasma and sedum) [90]; Peak at 53.42 ± 1.60 nm (urine) [91]	UC < EXQ (cell medium) [90]; UC < UF (cell medium) [57]; UC < TEI (cell medium and plasma) [89]; UC < UF, SEC < EXQ = TEI = PEG (plasma) [66]; SEC < SEC + DG = UC < EXQ (urine) [39]; UC < HFD (urine) [60]; EXQ, UCsuc < UF = UC < EXQmodif (urine) [67]	HSP70 and flotillin-1: UC = UF; TSG101: UC < UF (cell medium) [57]; CD9: UC = EXQ (cell medium) [90]; CD9, CD63 CD81, TSG101 (cell culture) [51]; CD81, CD63, CD9, TSG101, annexin 5 (cell culture and plasma) [89]; CD63: EXQ = TEI = PEG = SEC =UF = UC; no CD81 and Tsg101 (plasma) [66]; Flotillin, CD63 (plasma and serum) [90]); Alix, flotillin-1, CD9: UC = EXQ < SEC = SEC + DG (urine) [39]; TSG101: UC < HFD (urine) [60]; AQP2 and neprilysin: UF (none) < UC < UC + SEC (urine) [92]; High levels of CD63, CD9, AQP2; medium, of CD10 and FLT1; and low, of AIP1 (urine) [68]; Alix and TSG101: EXQ < UF < UC = UCsucr = EXQmodif (urine) [67]	Purity (ratio of EV to protein amounts): UC < UCsucr (cell culture) [93]; Amount: UC < EXQ (cell medium) [90]; Amount: UC, UCsucr < EXQmodif, EXQ, UF; purity: UF < EXQ < EXQmodif < UC, UCsucr (urine) [67]; SEC < SEC + ODG < DUC = EXQ (urine) [39]; Albumin: UC < UF; THP: UC = UF (urine) [70]; Albumin: UC + SEC < UF, UC (urine) [92]; THP: TEI < UC; lectin < Norgen (urine) [68]; Proteins: UC < EpCAM < SEC < EXQ (ascites) [40]	Peaks at 160–189 and 300–400 nt (cell culture) [51]; Amount: UC < TEI, mainly 200 nt and certain share of longer ones, including 18S and 28S rRNA (cell couture and plasma) [89]; Amount of sRNA: UC < Norgen; high rate of tRNA (plasma); sRNA: NOR = UC (serum) [90]; Lectin < UC, TEI < Norgen (2.7 ng/ml urine); ACTB1, SOD1, GNB2L1, GAPDH, EEF1A1, RPL6 transcripts: UC, TEI, lectin < Norgen (urine) [68]; Amount and purity: EXQ = UF < UC = UCsucr < EXQmodif (urine) [67]; Purity: UC < SEC = EpCAM < EXQ; Amount: UC, EpCAM = SEC < EXQ (ascites) [40]	Amount: UC < TEI (cell culture and plasma) [89]; Amount of miRNA: Norgen = UC (plasma and serum) [90]; Amount and purity; UF < EXQ = UC = UCsucr < EXQmodif (urine) [67]
(1.2) Sucrose density gradient ultracentrifugation (UCsucr)	104 ± 9.85 (cell medium) [93]; 20–100 nm (urine) [94]	UCsucr < PROSPR (plasma) [73]; EXQ, UCsucr < UF = UC < EXQmodif (urine) [67]	CD9, CD63, Alix, CD81: UCsucr < PROSPR (plasma) [73]; Alix and TSG101: EXQ (none) < UF < UC = UCsucr = EXQmodif (urine) [67]; Alix, CD9, TSG101, HSP70, AQP2 (urine) [94]	Purity (EV to protein amounts): UC < UCsucr (cell culture) [93]; Albumin and other blood plasma proteins: PROSPR < UCsucr (plasma) [73]; Amount: UC, UCsucr < EXQmodif, EXQ, UF; Purity: UF < EXQ < EXQmodif < UC, UCsucr (urine) [67]; THP (urine) [58]	Amount and purity: EXQ = UF < UC = UCsucr < EXOmodif (urine) [67]	Amount and purity: UF < EXQ = UC = UCsucr < EXQmodif (urine) [67]
(1.3) Iodixanol density gradient ultracentrifugation (DG)	50–100 nm (cell medium) [36]; EXQ = UF = SEC + particles > 200 nm; Unlike DG, where they are absent (cell medium) [57]	UC, DG < EpCAM (cell medium) [36]; DG < SEC < EXQ, UF (cell medium) [57]	TSG101, FAM125B, Rab11B, tetraspanin 8, proteins TFRC, PTGFRN: UC < DG; Alix, TSG101, HSP70: UC, DG < EpCAM (cell culture) [36]; HSP70, flotillin-1, TSG101: EXQ < UF < SEC, DG (cell medium) [57]	Purity (ratio per 1 μg protein): UF, EXQ (coisolation of non-EV protein) < DG (loss of sample) < SEC (cell medium) [57]
(2.1) Ultrafiltration (UF)	50–250 nm (cell medium) [57]; Peak of 70.57 ± 4.73 nm (urine) [91]; UC = UF (urine) peak of 115 ± 3 nm [58]	UC < UF (cell medium) [57]; UC < UF, SEC < EXQ = TEI = PEG (plasma) [66]; SEC < UF, EXQ (plasma) [57]; EXQ, UCsuc < UF = UC < EXQmodif (urine) [67]	HSP70 and flotillin-1: UC = UF; TSG101: UC < UF (cell medium) [57]; Flotillin-1: EXQ, UF < SEC (plasma) [57]; CD63: EXQ = TEI = PEG = SEC = UF = UC (plasma) [66]; CD24 and AQP2: UF < UC (urine) [58]; AQP2 and neprilysin: UF (none) < UC < UC + SEC (urine) [92]; EXQ < UF < UC = UCsucr = EXQmodif (urine) [67]	Albumin: SEC < UF, EXQ (plasma) [57]; Amount: UC, UCsucr < EXQmodif EXQ, UF; Purity: UF < EXQ < EXQmodif < UC, UCsucr (urine) [67]; Albumin: UC < UF; THP: UC = UF (urine) [58]; Albumin: UC + SEC < UF, UC (urine) [92]	Amount and purity: EXQ = UF < UC = UCsucr < EXOmodif (urine) [67]	Amount and purity: EXQ = UF < UC = UCsucr < EXOmodif (urine) [67]
(2.2) Hydrostatic dialysis (HFD) (urine) [60]	50–90 nm	UC < HFD	TSG101: UC < HFD	Most soluble proteins and THP are absent	Enrichment < 1000 nt; rRNA is absent	miRNA (10–40 nt) is present in sRNA (6–150 nt) fraction
(2.3) Size-exclusive chromatography (SEC)	EXQ = UF = SEC + particles of >200 nm unlike DG, where they are absent (cell medium) [57]; 80–200 nm (plasma) [42]; 70–500 nm (platelet-free plasma) [63]; SEC = EXQ = UF= 50–200 nm, mainly 50–100 nm (plasma) [57]; On the average, 246 ± 69 nm (urine) and 250 ± 28 nm (concentrated urine) [64]	DG < SEC < EXQ, UF (cell medium) [57]; UC < UF, SEC < EXQ = TEI = PEG (plasma) [66]; SEC < PEG, PROSPR (plasma) [42]; UC < SEC (ascites) [40]; SEC < SEC + DG = UC < EXQ (urine) [39]; SEC < UF, EXQ (plasma) [57]	HSP70, flotillin-1, TSG101: EXQ < UF < SEC, DG (cell medium) [57]; CD63 and CD9 (plasma) [63]; Flotillin-1: EXQ, UF < SEC (plasma) [57]; Flotillin-1 is present and calnexin is not (plasma) [57]; CD63: EXQ = TEI = PEG = SEC = UF = UC (plasma) [66]; CD63 and CD9 (urine) [64]; Alix, flotillin-1, CD9: UC = EXQ < SEC = SEC + DG (urine) [39]; AQP2 and neprilysin: UF (none) < UC < UC + SEC (urine) [92]	Purity (ratio per 1 μg protein): UF, EXQ (coisolation of non-EV protein) < DG (loss of sample) < SEC (cell medium) [57]; Albumin: SEC < UF, EXQ (plasma) [57]; Amount: SEC < PROSPR < PEG (plasma) [42]; SEC < SEC + DG < DG = EXQ (urine) [39]; Albumin: UC + SEC < UF, UC (urine) [92]; THP and albumin are absent (urine) [64]; Protein amount: UC < EpCAM < SEC < EXQ (ascites) [40]	Purity: UC < SEC = EpCAM < EXQ; Amount: UC, EpCAM = SEC < EXQ (ascites) [40]
(3.1) Polyethylene glycol (PEG)	Average size: EXQ = TEI = PEG (plasma) [66]; 80–200 nm; average size, 140 nm (plasma) [42]	UC < UF, SEC < EXQ = TEI = PEG (plasma) [66]; SEC < PEG, PROSPR (plasma) [42]	CD81 and Tsg101 (plasma); CD63: EXQ = TEI = PEG = SEC = UF = UC (plasma) [66]; LGALS3BP, CD5L, CD9 (plasma) [42]	Amount: SEC < PROSPR < PEG (plasma) [42]		Expression of eight miRNAs: TEI = EXQ = PEG (plasma) [66]
(3.2) TEI	Average size: EXQ = TEI = PEG (plasma) [66]; Mainly 30–150 nm, <300 nm (cell culture and plasma) [89]	UC < TEI (cell culture and plasma) [89]; UC < UF, SEC < EXQ = TEI = PEG (plasma) [66]	CD81, CD63, CD9, TSG101, annexin 5 (cell culture and plasma) [89]; CD81 and TSG101; CD63: EXQ = TEI = PEG = SEC = UF = UC (plasma) [66]; CD9, CD10, CD26, CD63, TSG101, CD10, AIP1/Alix, AQP2, FLT1: high level of CD26 and CD63; low level of AQP2 and FLT1 (urine) [68]	THP: TEI < UC, lectin < Norgen (urine) [68]	Amount: UC < TEI, mainly 200 nt and certain share of longer ones, including 18S and 28S rRNA (cell culture and plasma) [89]; Lectin < UC, TEI < Norgen (2.7 ng/ml urine); ACTB1, SOD1, GNB2L1, GAPDH, EEF1A1, RPL6 transcripts: UC, TEI, lectin < Norgen (urine) [68]	Expression of eight miRNAs: TEI = EXQ = PEG (plasma) [66]; Amount: UC < TEI (cell culture and plasma) [89]; miR-21 and miR-375: Norgen < TEI (urine) [95]
(3.3) ExoQuick (EXQ)	92.8 ± 13.8 and 78.0 ± 7.1 nm (cell medium) [90]; EXQ = UF = SEC + particles of >200 nm; Unlike DG, where they are absent (cell medium) [57]; SEC = EXQ = UF = 50–200 nm; mainly, 50–100 nm (plasma) [57]; Average size: EXQ = TEI = PEG (plasma) [66]; Peak of 83.80 ± 3.67 nm (urine) [91]	UC < EXQ (cell medium) [90]; DG < SEC < EXQ, UF (cell medium) [57]; SEC < UF, EXQ (plasma) [57]; UC < UF, SEC < EXQ = TEI = PEG (plasma) [66]; EXQ, UCsucr < UF = UC < EXQmodif (urine) [67]; SEC < SEC + DG = UC < EQ (urine) [39]	CD9: UC = EXQ (cell medium) [90]; HSP70, flotillin-1, TSG101: EXQ < UF < SEC, DG (cell medium) [57]; Flotillin-1: EXQ, UF < SEC (plasma) [57]; Flotillin-1 and calnexin are absent (plasma) [57]; CD81 and TSG101; CD63: EXQ = TEI = PEG = SEC = UF = UC (plasma) [66]; Alix, flotillin-1, CD9: UC = EXQ < SEC = SEC + DG (urine) [39]; Alix and TSG101: EXQ < UF < UC = UCsucr = EXQmodif (urine) [67]	Amount: UC < EXQ (cell medium) [90]; Purity (ratio per 1 μg protein): UF, EXQ (coisolation of non-EV protein) < DG (loss of sample) < SEC (cell medium) [57]; Albumin: SEC < UF, EXQ (plasma) [57]; Amount: UC, UCsucr < EXQmodif, EXQ, UF; purity: UF < EXQ < EXQmodif < UC, UCsucr (urine) [67]; SEC < SEC + DG < UC = EXQ (urine) [39]; Amount of proteins: UC < EpCAM < SEC < EXQ (ascites) [40]	Amount and purity: EXQ = UF < UC = UCsucr < EXQmodif (urine) [67]; Purity: UC < SEC = EpCAM < EXQ; Amount: UC, EpCAM = SEC < EXQ (ascites) [40]	Expression of eight miRNAs: TEI = EXQ = PEG (plasma) [66]; Amount and purity: UF < EXQ = UC = UCsucr < EXQmodif (urine) [67]; miRNA amount: EpCAM < EXQ (ascites) [40]
(3.4) Norgen			High levels of AIP1 and CD26; medium, of CD10, FLT1, CD9, TSG101; and low, of AQP2 and CD63 (urine) [68]	THP: TEI < UC, lectin < Norgen (urine) [68]	Amount of sRNA: UC < Norgen; high rate of tRNA (plasma) [90]; sRNA: NOR = UC (serum) [90]; Lectin < UC, TEI < Norgen (2.7 ng/ml urine); ACTB1, SOD1, GNB2L1, GAPDH, EEF1A1, RPL6 transcripts: UC, TEI, lectin < Norgen (urine) [68]	Amount of miRNA: Norgen = UC (plasma and serum) [90]; miR-21 and miR-375: Norgen < TEI (urine) [95]
(3.5) Precipitation with protamine		Protamine = UC (cell culture) [43]	CD63, CD9, CD81: protamine = UC (cell culture) [43]		Amount: protamine = UC (cell culture) [43]	Amount: protamine = UC (cell culture) [43]
(3.6) Precipitation with sodium acetate (NaAc)	NaAc = UC (cell culture) [44]		Alix and HSP70: NaAc = UC (cell culture) [44]	Amount: UC < NaAc, contamination with no-EV proteins (cell culture) [44]
(3.7) Precipitation of proteins with organic solvent (PROSPR)	PROSPR = UCsucr = 20–300 nm + aggregates of vesicles of different sizes + nonmembrane particles of 30 nm 30 nm (plasma) [73]; 74% particles ≤ 100 nm; 8.5% particles < 300 nm; 3.5%, < 500 nm; and 13%, >500 nm (CNS) [73]	UCsucr < PROSPR (plasma) [73]; SEC < PEG, PROSPR (plasma) [42]	CD9, CD63, Alix, CD81: UCsucr < PROSPR (plasma) [73]; CD9, CD63, CD81, LGALS3BP, CD5L are undetectable (plasma) [42]	Amount: SEC < PROSPR < PEG (plasma) [42]; Albumin and other blood plasma proteins: PROSPR < UCsucr (plasma) [73]; Amount of vesicular proteins is 7.2-fold larger and of exosomal proteins, 6.9-fold larger as compared with UCsucr (CNS) [73]
(4) Two-phase isolation (PEG + DEX)	PEG + DEX = UC (mixture of exosomes and proteins) [74, 75]	UC < PEG + DEX (mixture of exosomes and proteins) [74]; UC < EXQ < PEG + DEX (mixture of exosomes and proteins) [75]	CD81: UC < PEG + DEX (mixture of exosomes and proteins) [74]; CD81, CD9, Alix: UC < EXQ < PEG + DEX (mixture of exosomes and proteins, mouse plasma) [75]		Amount: Melan A and GAPDH, UC < EXQ < PEG + DEX (mixture of exosomes and proteins, mouse plasma) [74, 75]
(5.1) Antibodies	40–150 nm (cell medium) [56]	Anti-EpCAM (CG336) antibodies ob magnetic beads > UC, DG (cell medium) [56]; UC, DG < EpCAM (cell medium) [36]	Alix, TSG101, HSP70 (cell medium) [56]	Anti-EpCAM (CG336) antibodies ob magnetic beads > DG > UC (cell medium) [56]; Amount of proteins: UC < EpCAM < SEC < EXQ (ascites) [40]	Purity: UC < SEC = EpCAM < EXQ; Amount: UC, EpCAM = SEC < EXQ (ascites) [40]	miRNA amount: EpCAM < EXQ (ascites) [40]
(5.2) Annexin (cell medium and blood plasma) [78]			EV fluorescent staining (Hoechst 3342 or pyronin Y)		Detection of B2M and CK19 transcripts (RT-PCR)
(5.3) Lectins		Lectin = UC (urine) [82]	High level of CD9; medium level of TSG101, AIP1/Alix, FLT1; low level of AIP1, CD10, CD26, AQP2, CD63 (urine) [82]	THP: TEI < UC, lectin < Norgen (urine) [82]; THP: TEI < UC, lectin < Norgen (urine) [68]	Lectin < UC, TEI < Norgen (2.7 ng/ml urine); ACTB1, SOD1, GNB2L1, GAPDH, EEF1A1, RPL6 transcripts: UC, TEI, lectin < Norgen (urine) [82]; Lectin < UC, TEI < Norgen (2.7 ng/ml urine); ACTB1, SOD1, GNB2L1, GAPDH, EEF1A1, RPL6 transcripts: UC, TEI, lectin < Norgen (urine) [68]
(5.4) Heparin (cell medium and blood plasma) [79]	Heparin sorbent = UC		Heparin sorbent = UC, ExoQuick-ТС	Heparin sorbent < UC	Heparin sorbent = UC, ExoQuick-ТС (GAPDH, EGFR, LINE1, RPL11, CD63, cMyc)
(5.5) Tim protein (cell medium) [77]	106 nm	Total Exosome Isolation Reagent > TIM4	TIM4 > Total Exosome Isolation Reagent, UC (CD63, CD9, CD81)	Total Exosome Isolation Reagent, UC > TIM4	TIM4 > UC (GAPDH)	TIM4 > UC (miR-16, miR-92a, miR-142-3p)
(5.6) Vn peptide (cell medium, blood plasma, urine) [80]	30–100 nm	Vn peptide = UCsucr	Vn peptide > UСsucr (CD9, CD63, CD24, HSP70, Alix)		Vn peptide = UСsucr, ExoQuick-TC	Vn peptide = UСsucr, ExoQuick-TCU
(6) Microfluidic devices	75 ± 15 nm (cell culture and blood plasma) [84]; 100 nm (blood serum and glioblastoma multiforme) [96]; 150 nm (whole blood) [85]		CD63 and EpCAM (cell culture and blood plasma) [84]; CD63 (blood serum and glioblastoma multiforme) [96]; CD9, CD63, CD81 (whole blood) [85]		GAPDH and IDH-1 (blood serum and glioblastoma multiforme) [96]; Purity (RNA/protein ratio): UC > microfluidic filtration system; RNA amount (Melan A): UC = microfluidic filtration system (whole blood) [85]
(7) KeepEX (urine) [88]	KeepEX = UC	KeepEX > UC	KeepEX > UC (CD9, PDX, CD59, CD63)		KeepEX = UC

UC, ultracentrifugation; UCsucr, sucrose density gradient ultracentrifugation; DG, iodixanol density gradient ultracentrifugation; UF, micro- and ultrafiltration; HFD, hydrostatic dialysis; SEC, size-exclusive chromatography; EV, precipitation with hydrophilic polymers; PEG, with polyethylene glycol; EXQ, using ExoQuick (System Bioscience, United States); TEI, using Total Exosome Isolation Kit (Invitrogen/ThermoFisher Scientific, United States); Norgen, Exosome RNA Isolation Kit (Norgen, Biotek Corp.); protamine, using EV precipitation with protamine; NaAc, using EV precipitation with NaAc; PROSPR, EV isolation via protein precipitation with organic solvent; PEG + DEX, distributive method; and EpCAM, isolation using antibodies to EpCAM.