The generation and use of recombinant extracellular vesicles as biological reference material

Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications.

of 0.01 detected in rEV and mock EV. (d) Venn diagram of proteins from the ESCRT pathway, and proteins detected in rEV and mock EV. (e) Comparison of the top 6 molecular functions and biological processes attributed to the proteins enriched in rEV and mock EV. Source data can be accessed at the PRIDE database (PXD010269).

Supplementary figure 5
Supplementary figure 5: Lipidome comparison of rEV and mock EV. Lipids were extracted from equal numbers of rEV and mock EV separated by ODG centrifugation from medium conditioned by respectively gag-EGFP transfected and mock transfected HEK293T cells and were subjected to ESI-MS/MS. (a) Log of relative expression values of all detected phospholipids in rEV and mock EV (n=3). (b) μg cholesterol per particle of rEV and mock EV separated by ODG centrifugation from medium conditioned by respectively gag-EGFP transfected and mock transfected HEK293T cells and sample EV separated by ODG centrifugation from medium conditioned by breast cancer cells (MCF7, 4T1) or cancer associated fibroblasts (CAF) or urine. (n=9 (rEV), n=6 (mock EV), n=3 (sample EV from MCF7, 4T1, CAF and urine)) data are presented as (mean, SD). Source data are provided as a source data file.

Supplementary figure 6
Supplementary figure 6: rEV separation by optiprep velocity gradient (OVG) centrifugation. Western blot analysis for gag-EGFP and EV-associated proteins ALIX, TSG101, flotillin-1, syntenin-1, CD81 and CD9 of (a) individual OVG fractions collected from top (1) to bottom (15) or (b) pooled OVG fractions from top (1-3) to bottom (14-15) obtained after OVG centrifugation of medium conditioned by gag-EGFP transfected HEK293T cells. (c) Percentage of fluorescent rEV (ratio fNTA/NTA) in individual OVG fractions and (d) size distributions of OVG separated rEV measured with NTA under both scatter and fluorescence mode. (e) TEM images of EV (OVG fractions 4-5, 6-7) and rEV (OVG fractions 10-13) obtained by OVG centrifugation of medium conditioned by gag-EGFP transfected HEK293T cells. Indirect quantification of rEV separated by OVG centrifugation from medium conditioned by gag-EGFP transfected HEK293T cells using (f) a fluorescent plate reader showing a linear correlation of the relative fluorescence units (RFU) and number of rEV, (g) an ELISA for p24 showing a linear correlation of p24 concentration and number of rEV and (h) RT-qPCR for EGFP mRNA showing a semi-logarithmic correlation with the Cq values and number of rEV. (i) Density distribution of OVG separated rEV spiked in PBS and proteinase K (PK)-treated plasma obtained by ODG centrifugation followed by fNTA measurement of ODG fractions (n=2). Data are presented as (mean, SD) (j) Proteomic comparison of endogenous EV (OVG fractions 4-5 and 6-7) and rEV (OVG fraction 10-13) separated by OVG centrifugation versus rEV separated by ODG centrifugation from medium conditioned by gag-EGFP transfected HEK293T. Source data are provided as a source data file.

Supplementary figure 7
Supplementary figure 7: Stability of rEV at -80 °C or 4 °C. The structural and biological stability of rEV was assessed in different storage conditions. (a) Relative rEV concentration and (b) size modus measured by fNTA immediately upon rEV separation by ODG centrifugation of medium conditioned by gag-EGFP transfected HEK293T cells (fresh) and after 6 months -80°C freeze followed by thaw or 12 months -80°C freeze followed by thaw. Data are presented as (mean, SD). (c) Relative rEV concentration and (d) size modus measured by fNTA of fresh rEV or after storage at 4 °C up to one week (n=3). (e) Density distribution of differently stored rEV (fresh, -80 °C or 4 °C) spiked in PBS obtained by ODG centrifugation followed by fNTA measurement of density fractions. (f) Western blot analysis for gag-EGFP and CD81 of -80 °C stored rEV spiked in PBS followed by immune precipitation (IP) with anti-CD81 coated magnetic beads together with the flow through (FT). Source data are provided as a source data file.

Supplementary figure 8
Supplementary figure 8: Lyophilization of rEV. rEV can be lyophilized in PBS supplemented with 5% trehalose without structural and biological changes. (a) Size distribution measured by fNTA of rEV before and after lyophilization (n=3). (b) Scatter plots showing detection of rEV with HR-FC before and after lyophilization (n=3). (c) TEM of rEV before (fresh) and after lyophilization. (d) Density distribution of rEV before (fresh) and after lyophilization in proteinase K (PK) treated plasma obtained by ODG centrifugation followed by fNTA measurement of ODG fractions. Source data are provided as a source data file.

Supplementary figure 11
Supplementary figure 11: Recovery efficiency calculation is independent from number of rEV spiked. rEV was spiked in plasma in varying numbers and separated by ODG centrifugation or SEC followed by recovery efficiency calculation with fNTA and ELISA respectively (n=2). Data is presented as (mean, SD). Source data are provided as a source data file.

Supplementary table 1
Supplementary table 1: rEV mitigate for inter-user variability. EV separation was performed in 6 technical replicates from PK treated plasma spiked with 1x10 10 rEV by ODG. Next, inter-user variation was induced by sample replacement with PBS (see material and methods). The total number of particles was measured using NTA in scatter mode. The total number of rEV was measured using fNTA. From the latter the separtion efficiency was calculated (knowing that 1x10 10 rEV were spiked) and implemented to normalize the number of scatter particles. Data is representative for figure 5 e.

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