Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples

Figures & data

Fig. 1.  Diagram of the isolation procedure and the initial analysis of the fractions.

Fig. 2.  Total protein concentration in SEC fractions.

Bradford assay results of non-concentrated (n=7, panel a) and concentrated (n=5, panel b) samples from healthy donors (HD). In both graphs, the x-axes show the collected fractions and the y-axes represent the protein concentration (mg/mL).

Fig. 3.  Urine EVs are eluted in low protein containing SEC fractions.

In panel a, a representative analyses of a SEC processed sample is shown. In each fraction (indicated on the x-axis), the expression of CD9 and CD63 and the total protein content were determined. The left axis shows the total protein content (mg/mL), whereas the right axis shows the median fluorescence intensity (MFI) data for CD9 and CD63. The isotype control for flow cytometry assay is depicted by a dotted line.

In panel b, fractions showing the highest MFI value for each CD marker (CD9 square symbols and CD63 triangle symbols) were grouped (n=10). Fractions in which protein content was first detected are also shown (circles, n=8). Higher CD9 and CD63 MFI values were routinely detected between fractions 7 and 10, well before any protein elution was detectable.

Table I. Size distribution and EVs concentration from NTA analyses

NTA	3 mL Urine (n=5)	120 mL Urine (n=4)
Mean size	246±69	250±28
Mode size	225±74	230±51
SD	88.5	76
Particles/mL	4.1E+08 (0.63–7.85)	4E+10 (0.09–8.01)

The size distribution and EVs concentration calculated from NTA experiments of tetraspanin-peak fractions are shown in this table. The number of particles is expressed as the mean of the different experiments. The range is indicated between brackets.

Fig. 4.  Analyses of the EVs obtained after SEC procedure by NTA.

Representative NTA analyses of the tetraspanin-peak fractions from HD4 (non-concentrated, left) and HD4C (concentrated, right) urine samples.

Fig. 5.  Analyses of the EVs obtained after SEC procedure by cryo-EM.

Panel a shows a cryo-EM of vesicles contained in a tetraspanin-peak fraction from non-concentrated (left) and concentrated (right) samples. Scale bar is 500 nm. Arrows pointing at EVs. In panel b, the size of these vesicles was measured (diameter in nm) from a set of images of non-concentrated (left, n=36 EVs from 10 images) and concentrated (right, n=63 EVs, from 17 images) samples using ImageJ software (NIH).

Table II. Summary of proteomic results

	Membrane proteins related to EVS	Cytosolic proteins related to EVs	Proteins related with other vesicles	Extracellular proteins	Urinary tract membrane proteins	Urinary tract soluble protein
Concentrated urine (100 mL)	LGALS3BP ATP6V1H GPRC5C GPRC5B	ANXA2 ANXA11 ANXA4 EZR SDCBP TSG101 ALIX	NAGLU MAN1A1	A2M TF ALB	ATPV1B1 ATP1V1A SLC12A3 MME^a ANPEP SCL12A1 UPK1A RHCG LRP2^a	NPHS2
Non-concentrated urine (3 mL)	GPRC5C GPRC5B	ALIX			SCL12A1
UC (Refs. 12,25) (200–300 mL)	LGALS3BP ATP6V1H GPRC5C GPRC5B	ANXA2 ANXA4 ANXA11 ALIX EZR SDCBP TSG101	NAGLU Apo-lipoproteins Mitochondria related ion transports	ALB TF	ATPV1B1 ATP1V1A SLC MME ANPEP UPK1A	NPHS2

A selected group of proteins (EV-related and non-EV-related) from the proteomic analysis of tetraspanin-peak fractions from concentrated or non-concentrated urine are shown. The table is organized based on the minimal requirements to characterize EVs as published (35) containing: transmembrane or lipid bound proteins related to EVs, cytosolic proteins related to EVs, proteins related to other vesicles (i.e. lysosomes, mitochondria) and extracellular proteins. In addition, a group of urinary-tract-related proteins have been also included. For comparison purposes, the results of 2 previously published studies are also indicated (UC).

LGALS3BP: galectin-3-binding protein; ATP6V1: V-type proton ATPase; GPRC5: G-protein-coupled receptor family C; ANXA: annexin; EZR: ezrin; SDCBP: isoform 3 of syntenin-1; TSG101: tumour susceptibility gene 101; ALIX: programmed cell death 6-interacting protein; NAGLU: α-N-acetylglucosaminidase; MAN1A1: mannosyl-oligosaccharide 1,2-α-mannosidase; ALB: albumin; TF: serotransferrin; A2M: α-2-macroglobulin; SLC: solute carrier family; MME: neprilysin; aminopeptidase N; UPK1a: uroplakin; RHCG: ammonium transporter Rh-type C; LRP2: low-density lipoprotein receptor-related protein; NPHS2: podocin.^aSpecific proteins belonging to the convoluted tubule epithelia (brush border).

Fig. 6.  EV fractions from concentrated urine are enriched in protein content.

Coomassie blue stained SDS–PAGE of the concentrate urine (lane C), the 17,000g pellet (lane 17,000g) and 7 fractions including the tetraspanin-peak fractions (F8–F12) and 2 later fractions (F19 and F20) are shown in panel a. Molecular weight is indicated in the first lane (MW). THP and albumin bands are indicated in the gel.

In panel b, silver staining of the same SDS–PAGE showing the protein content of tetraspanin-peak fractions. THP and albumin bands are also indicated.

Fig. 7.  Analyses of the RNA content of SEC fractions.

Panel a shows a representative (n=3) bioanalyser profile of small RNA of a single tetraspanin-peak fraction obtained from a non-concentrated sample, HD5. In panel b, bioanalyser profile of a concentrated, HD5C, sample is shown.

Lötvall J, Hill AF, Hochberg F, Buzás EI, Di Vizio D, Gardiner C et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014; 3: 26913. doi: http://dx.doi.org/10.3402/jev.v3.26913.

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