Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.


Age segregated by gender
Supplementary Table S2.Data corresponding to ultracentrifugation time period optimization (UC25min, UC48min and UC60min protocols).Average values from total particle recovery, particle concentration, particle size mean and standard deviations from Nanoparticle Tracking Analysis.Average values from total protein recovery and protein concentration (evaluated using microBCA), purity ratio and particle number in 20 µL of EV sample loaded for Western-Blot are represented values based on 7 independent sample replicates.
Supplementary Table S8.Information about characterization from SEVs isolated from 7 patients.
and Alix are represented in different samples.THP (urine contaminant), Lamin A/C and Cytochrome C (associated with intracellular compartments) markers are also represented.PC3 cell line lysate was used as a positive control for Lamin A/C and Cytochrome C markers.images were cropped; the original blots are presented in Figure S10 from Supplementary Material 2. (c) Dot plots of particle concentration, particle mean size, protein concentration and purity ratio results for all samples used among the tested conditions.Horizontal black lines represent the means of tested samples per condition.All parameters were measured in 3 samples for each tested method.
´ Supplementary Figure S4.Graphical representations of Nanoparticle Tracking Analysis regarding the comparisons of UC48min protocol without any treatment, with 2-mecarptoethanol treatment and with NaCl treatment.Nanoparticle tracking analysis (NTA) graphs display in y-axis: concentration (Particle/mL), and in x-axis: size (nm).Experiments were conducted in 3 independent patients.

Table S3 .
Total particle recovery, particle concentration particle size mean, and respective standard deviations measured by Nanoparticle Tracking Analysis are represented.Total protein recovery, protein concentration (microBCA) and purity ratio values (calculated by dividing particle concentration by protein concentration) are also described.Particle number in 20 µL of sample loaded for Western-Blot is represented.All measurements were based on 3 independent experimental replicates.Information corresponding to large EVs (LEVs) pelleting (UCLEVs) versus urine supernatant filtering (UC48min).
Total particle recovery, particle concentration, particle size mean with respective standard deviations obtained by Nanoparticle Tracking Analysis.Total protein recovery and protein concentration measured using microBCA.Purity ratio and particle number in 20 µL of EV sample (loaded in Western-blot) are also represented.Values were based on 3 independent samples.Values from total particle recovery, particle concentration, particle size mean and respective standard deviations measured using Nanoparticle Tracking Analysis.Total protein recovery and protein

Table S4 .
Information about characterization from SEVs isolated without washing step (UC48min) and with washing step (UCwash).concentration(assessedbymicroBCA),purity ratio and particle number in 20 µL of sample loaded in Western-Blot are represented.Values were based on 3 independent experimental replicates.Supplementary TableS5.Information of characterization from SEVs isolated with optimized differential ultracentrifugation (UC48min), density ultracentrifugation (dUC) and Exoquick (EXO).Values from total particle recovery, particle concentration, particle size mean with standard deviations from Nanoparticle Tracking Analysis.Total protein recovery, protein concentration (measured by microBCA), purity ratio and particle number in 20 µL of EV sample used for Western-Blot are represented.Values were based on 3 independent samples.

Table S6 .
Information about characterization from SEVs isolated from 3 patients by UC48min and dUC methods.Average values from total particle recovery, particle concentration, particle size mean and standard deviations from Nanoparticle Tracking Analysis.Average values from total protein recovery and protein concentration (evaluated using microBCA), purity ratio and particle number in 20 µL of EV sample loaded for Western-Blot are represented.Values are based on 3 independent sample replicates.Supplementary TableS7.Information about RNA concentration obtained by UC48min and dUC method under RNAse and without RNAse conditions.