SEC was effective in removing plasma proteins and HDL but not low-density lipoproteins.

(A) 500 μl of plasma was loaded on a SEC column and PFs were collected. (B) Cryo-EM images of PFs obtained from the plasma of 5 healthy individuals. Vesicles could be easily identified and the images revealed a clean background suggesting minimum protein contamination. Moreover, HDL was sparse in the Cryo-EM images, indicating that they were also largely removed. Furthermore, sEVs, having clearly defined bilayer (red arrows), were easily distinguishable from lipoproteins (representative particles marked by yellow arrows). The ratio of counted sEVs to all vesicles are shown under each image. All scale bars represent 200 nm.

Design of a density gradient setup in a small volume format.

(A) 500 μl PBS was overlaid on 800 μl 10% iodixanol solution and 20 μl 50% iodixanol cushion. The 1.5 ml tubes were subjected to ultracentrifugation using a fixed-angle rotor at an average speed of 135,000 g for 2, 6 and 16 h at 4 °C to establish a density gradient profile. (B) Density gradient profiles along the 1.5 ml tubes after ultracentrifugation. The 2 h spinning time gave a density profile with the largest separation zone of 1.05~1.08 g/ml and smallest sEV zone of >1.08 g/ml. The data shows the average ± standard deviation of 7 repeats for 2 h, 3 repeats each for 6 and 16 h.

Particle and protein concentrations along the 1.5 ml tube after applying DGUC to plasma.

(A) The 1.5 ml tube was fractionated into 13 fractions. (B) Particle concentration ± std measured by NTA of different fractions (n=3). (C) Protein concentrations ± std measured by BCA of different fractions (n=3).

Schematic representation of the SEC-DGUC and DGUC-SEC protocols.

(A) Illustrates the SEC-DGUC protocol sequence. Starting from the left, SEC is used initially to separate plasma proteins and HDL. Subsequently, DGUC is employed to separate low-density lipoproteins (i.e., IDL, VLDL, LDL) from sEVs. (B) Depicts the DGUC-SEC protocol, wherein DGUC is initially employed to segregate high-density components from those of lower density, before SEC is applied to partition any remaining proteins from the sEVs.

DGUC in the 1.5 ml tube format effectively separated sEVs from lipoproteins.

PFs obtained from SEC were concentrated into 500 μl, loaded onto the density gradient and subjected to ultracentrifugation as previously described. After DGUC, the 1.5 ml tube was fractionated into 13 fractions, which were each examined for their particle concentration (by NTA) and the presence of sEVs and lipoproteins (by TEM). In the sEV zone (bottom of the tube), where density was higher than 1.08 g/ml, high purity sEVs were indeed observed. A mixed population of sEVs and lipoproteins was observed within the density zone of 1.05~1.08 g/ml. Interestingly, the particle numbers in this density zone were low, thus creating an effective separation zone between lipoproteins and sEVs. The RNA profiles of corresponding particle populations are shown on the right. All scale bars represent 200 nm.

The high purity of sEVs in SEC-DGUC-1 was demonstrated by WB and Cryo-EM.

(A) The 1.5 ml tube subjected to the SEC-DGUC protocol was fractionated into 4 fractions for easier analysis. (B) Western blot analyses of original plasma, SEC-PF, SEC-DGUC-1 to 4 using sEV and lipoprotein markers. (C) & (D) Cryo-EM images showing the comparison between SEC-PF and SEC-DGUC-1. The SEC-DGUC-1 in (D) was obtained from non-fasting plasma collected in EDTA tubes. All scale bars represent 200 nm.

Comparison of sEVs isolated by SEC-DGUC and DGUC-SEC protocols.

(A) Western blot of SEC-DGUC-1, DGUC-SEC-PF, sEV isolated by dUC obtained from 2 ml of plasma of the same source. (B) Particle size distributions in SEC-DGUC-1 and DGUC-SEC-PF measured by TEM. (C) and (D) TEM images of SEC-DGUC-1 and DGUC-SEC-PF. (E) and (F) Total RNA analyses of SEC-DGUC-1 and DGUC-SEC-PF. Note that data in (E) is the same data shown in the bottom right subfigure of Figure 5.

Flow cytometry assay using MACSPlex exosome kit to evaluate sEV isolates.

(A) Illustration of the principle of MACSPlex exosome kit. 37 types of beads with different fluorescent colors are functionalized with specific antibodies against sEV surface proteins. sEVs captured by the beads are detected with flow cytometry using APC labeled anti-CD9, CD63, CD81 antibodies. (B) Flow cytometry gating setup. (C) A representative data showing the comparison of median fluorescence intensity (MFI) ± standard error of the mean (SEM) from sEV isolates obtained from SEC-PF and SEC-DGUC-1. Equal numbers of particles (5×108, based on NTA) were loaded for both samples. (D) Comparison of signal patterns among plasma, SEC-DGUC-1, SEC-PF and dUC. The Results showed that sEVs isolated from dUC deviate from plasma whereas, sEVs in the SEC-PF and SEC-DGUC-1 mirror the sEV population in plasma.

Functional annotation of proteins identified by LC-MS/MS.

(A) Functional annotation of proteins identified in SEC-DGUC-1. (B) Functional annotation of proteins identified in SEC-PF.

Repeatability of the SEC-DGUC protocol.

(A) Particle concentration profiles (by NTA) along the 1.5 ml tube from 4 technical replicates subjected to SEC-DGUC protocol. (B) Particle size distributions measured by NTA in the four fractions indicated on the left figure. Four technical replicates subjected to SEC-DGUC protocol were shown.

Reliability of the SEC-DGUC protocol.

TEM images of SEC-DGUC-1 were obtained from 5 fasting plasma (Biobank samples, EDTA tubes). High purity of sEVs represented by the low-contrast, cup-shaped vesicles was evident across different samples, suggesting the SEC-DGUC was reliable in obtaining high-purity sEVs. All scale bars represent 200 nm.

Particle numbers in different steps during the SEC-DGUC protocol, calculated based on NTA measurement.

The first column provides the total particle numbers measured in 500 μl fasting plasma (~1 ml of whole blood) from 5 individuals. The second column provides the corresponding total vesicle numbers in SEC-PF after the plasma was subjected to SEC. The third column provides the corresponding total vesicle numbers in SEC-DGUC-1 after the SEC-PF was subjected to DGUC.

SEC elution profiles, EM images of the PF and particle size distributions.

(A) SEC elution profiles according to particle concentration (by NTA) of 6 different plasma sources (left) and 4 repeats of the same plasma source (middle). Protein concentrations (by BCA, right) in corresponding to the elution profiles of the 6 different plasma sources shown on the left. 500 μl of plasma was loaded onto the SEC column and 10 fractions of 500 μl each were collected and analyzed. Fraction 7 to 10 were pooled to constitute the SEC-PF because of their high particle concentrations and low protein concentrations. (B) Representative TEM images of the PF. (C) Cryo-EM images of the SEC-PF. (D), (E) Typical particle size distributions of a plasma and SEC-PF measured by NTA. (E) A histogram of particle diameter obtained from the TEM images shown in (B).

Distribution of sEVs and lipoproteins within the density gradient after DGUC.

(A) SEC-PFs (6 ml) were placed on top of a density gradient cushion constructed with 2 ml 10%, 2 ml 30%, and 2 ml 50% iodixanol solutions and centrifuged at 150,000 × g for 2 h at 4 °C. (B) After DGUC, the tube was fractionated into 42 fractions, which were each examined for their densities, particle concentrations (by NTA) and presence of sEVs and lipoproteins (by TEM). The dominance of lipoproteins was evident in fractions of density <1.05 g/ml, which is designated as LP zone. sEVs started to appear when density exceeded 1.05 g/ml but a significant level of lipoproteins was present until the density of 1.08 g/ml. Therefore, the density region of 1.05~1.08 g/ml was designated as LP+sEV zone. Beyond 1.08 g/ml, lipoproteins diminished drastically and the density region of >1.08 g/ml was designated as the sEV zone. All scale bars represent 200 nm.

TEM images and size distributions of the 13 fractions collected from the 1.5 ml tube following SEC-DGUC.

(A) Representative TEM images of each of the 13 fractions. SEC-DGUC-1 clearly showed minimum presence of lipoprotein (high contrast lighter color particles) compared to other fractions. Moreover, the presence of sEVs (low contrast and cup-shaped particles) is evident in SEC-DGUC-1. (B) Particle size distributions measured according to the TEM images of the 13 fractions. Note the differences in the particle size distributions obtained from NTA measurement and TEM.

(A) The 1.5 ml tube was fractionated into 13 fractions. (B) Size distributions (by NTA) of the 13 fractions collected from the 1.5 ml tube following SEC-DGUC protocol. The particle size distribution in SEC-DGUC-1 was distinct from the rest of the fractions. Even in SEC-DGUC-2 (highlighted in purple), the size distribution follows fractions 3~13, implying the dominance of lipoproteins in these fractions. (C) The 1.5 ml tube was fractionated into 4 fractions in order to examine if 2 h spinning time was sufficient to isolate sEV from lipoproteins in the 1.5 ml tube format DGUC. (D) The resulting size distributions of SEC-DGUC-1s (by NTA) were highly similar for 2 h and 16 h spinning time. (E) The particle concentrations of the four individual fractions along the 1.5 ml tube closely resembled each other for 2 h and 16 h, implying that there were no significant loss of sEV for 2 h spinning time in comparison to 16 h.

Additional Cryo-EM images of SEC-DGUC-1.

Red arrows represent sEVs and yellow arrows represent typical lipoproteins. The SEC-DGUC-1 was obtained from non-fasting plasma collected in EDTA tubes. All scale bars represent 200 nm.

Flow cytometry data of SEC-PF vs. SEC-DGUC-1 obtained from two plasma sources using MACSPlex exosome kit.

Data from two plasma sources demonstrated a similar trend of much stronger signals of SEC-DGUC-1 compared to SEC-PF in flow cytometry using MACSPlex exosome kit. Data are Median ± SEM.

Repeatability of SEC.

(A-C) Particles size distributions of SEC-PF measured by NTA. (A), (B) and (C) correspond to the first, second and third rows of the table, respectively. The highly overlapped size distributions allude to the consistency in the particles eluted from SEC columns, even though the number of particles eluted varied up to 55%. The table lists six experiments with various numbers of repeats of SEC. The average particle concentrations, standard deviation and CV (SD/average ratio) are listed together with the number of measurements made.

Repeatability of SEC-DGUC protocol.

NTA measurements of two experiments to test repeatability after SEC-DGUC. The figure displays the size distributions measured by NTA of the experiment listed in the second row of the table. The size distributions of the experiment listed in the first row of the table were presented in main Figure 9. The table lists the average particle concentrations of SEC-DGUC-1 and the standard deviation, CV (SD/average ratio) together with the number of measurements made.

Reliability of particle concentration profiles along the 1.5 ml tube (by NTA).

(A) Detailed particle concentration profiles (13 fractions) of four different samples along with the calibrated density profile shown in Figure 2. (B) Five particle concentration profiles (4 fractions shown in Figure 9B) in SEC-DGUC-1 corresponding to the biobank plasma samples (EDTA tubes) shown in Figure 11. Note that the data from (A) and (B) are from different plasma sources.

Size distributions (by NTA) of SEC-DGUC-1 obtained from 5 fasting plasma corresponding to

Figure 11.

The particle size distributions did not overlap, reflecting the variation of sEV population among different plasma sources.