Standardized procedure to measure the size distribution of extracellular vesicles together with other particles in biofluids with microfluidic resistive pulse sensing

Abstract

The particle size distribution (PSD) of extracellular vesicles (EVs) and other submicron particles in biofluids is commonly measured by nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). A new technique for measuring the PSD is microfluidic resistive pulse sensing (MRPS). Because specific guidelines for measuring EVs together with other particles in biofluids with MRPS are lacking, we developed an operating procedure to reproducibly measure the PSD. The PSDs of particles in human plasma, conditioned medium of PC3 prostate cancer cell line (PC3 CM), and human urine were measured with MRPS (nCS1, Spectradyne LLC) to investigate: (i) the optimal diluent that reduces the interfacial tension of the sample while keeping EVs intact, (ii) the lower limit of detection (LoD) of particle size, (iii) the reproducibility of the PSD, (iv) the optimal dilution for measuring the PSD, and (v) the agreement in measured concentration between microfluidic cartridges with overlapping detection ranges. We found that the optimal diluent is 0.1% bovine serum albumin (w/v) in Dulbecco’s phosphate-buffered saline. Based on the shape of the PSD, which is expected to follow a power-law function within the full detection range, we obtained a lower LoD of 75 nm for plasma and PC3 CM and 65 nm for urine. Normalized PSDs are reproducible (R2 > 0.950) at dilutions between 10–100x for plasma, 5–20x for PC3 CM, and 2–4x for urine. Furthermore, sample dilution does not impact the dilution-corrected concentration when the microfluidic cartridges are operated within their specified concentration ranges. PSDs from microfluidic cartridges with overlapping detection ranges agreed well (R2 > 0.936) and when combined the overall PSD spanned 5 orders of magnitude of measured concentration. Based on these findings, we have developed operating guidelines to reproducibly measure the PSD of EVs together with other particles in biofluids with MRPS.

Tween-20 Induces the Structural Remodeling of Single Lipid Vesicles

“…Instead, this observation implies a structural change, namely expansion, taking place within individual vesicles, with minimal loss of lipid material to solution. This result is opposed to previous observations where Tween-20 had no effect on the modal size distribution of human-derived extracellular vesicles (EVs); 50 however, an explanation for this discrepancy rests in the lipid composition. In this work we used model membranes composed of POPC lipids, whereas EVs are enriched in cholesterol.…”

Tween-20 induces the structural remodelling of single lipid vesicles

“…Instead, this observation points towards a substantial structural change, namely swelling, taking place within individual surface-immobilized vesicles, with minimal loss of lipid material to the solution. Notably, this result is opposed to previous observations where Tween-20 had no effect on the modal size distribution of human-derived extracellular vesicles (EVs) (45). An explanation for this discrepancy rests in the lipid composition; while in this work we used highly-controllable model-membranes composed primarily of POPC lipids, most studies indicate that EVs are enriched in cholesterol compared with the total lipid pool(46).…”

“…Even if only a single calcium ion then enters the vesicle, the local Ca 2+ concentration increases by several hundred nM, which is readily detectable via the Cal-520 signal enhancement assay(33). It is of interest to note that this scenario, while consistent with recent work suggesting Tween-20 induces bulging of live cells(4), differs from those observed using extracellular vesicles, where the vesicle concentration reduced gradually with increasing Tween-20, but without variation in particle size(45, 54). In the latter case, the concentration of highly curved extracellular vesicles (EVs) with a similar size distribution to those used here, was strongly modulated by Tween-20, especially at detergent concentrations above the cmc, with the observed concentration reduction attributed to either large vesicle aggregates breaking apart or the lysis of single EVs.…”

Storage conditions determine the characteristics of red blood cell derived extracellular vesicles

“…MRPS measurements were carried out using factory calibrated TS-400 microfluidic cartridges with a measurement range from 65 to 400 nm. REV samples were diluted with filtered (Amicon Ultra 0.5 mL MWCO100 kDa membrane filter, Merck Millipore, Germany) bovine serum albumin (BSA, Sigma-Aldrich, Hungary) solution in PBS buffer (pH = 7.4, Sigma-Aldrich, Hungary) 40 , 60 . The addition of BSA solution was adjusted to the REV concentration as follows: REV_3days:BSA (10 mg/mL) = 9:1 volume ratio; REV_8days:BSA (1 mg/mL) = 1:9 volume ratio and REV_21days:BSA (1 mg/mL) 1:9 volume ratio.…”