Multiparametric analysis of small extracellular vesicles purified by a rapid and label-free lab on a chip device

Introduction:: Conventional purification methods of small extracellular vesicles (sEVs) suffer from significant shortcomings including low purity, low capture efficiency, long processing times, large sample volume requirement, need for specialized equipment and trained personnel, and high costs. Our group has previously developed a label-free insulator-based dielectrophoretic (iDEP) device for rapid and selective entrapment of sEVs based on their unique dielectric properties and size^1-3. Here we report a comprehensive three-fold characterization of sEVs isolated using the iDEP device from human biofluids including serum, plasma, and urine by utilizing conventional flow cytometry (cFCM), advanced imaging flow cytometry (iFCM), and next generation miRNA sequencing indicating high yield and purity.


Materials and Methods:: Serum (n=6), plasma (n=2), and urine (n=3) samples from genitourinary tract cancer cases were obtained from the University of Cincinnati Biorepository (IRB#2015-2364). The device was fabricated as reported previously³. sEVs were isolated from biofluids using the iDEP device and analyzed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). For cFCM and iFCM, sEVs were stained with CD63-FITC (H5C6, 353005), CD81-APC (5A6, 349509), and Isotype-IgG1 FITC

(MOPC21, 400109) from BioLegend. cFCM was performed using a Cytek Aurora cytometer and analysis was performed using FlowJo. Data were collected for 100 μL/sample with flow rate at low and threshold count at 5000. iFCM was performed for extracted sEVs using ImageStreamX Mark II and analyzed using IDEAS. Data acquisition was performed with fluidics set to low, sensitivity high, core size 7 μm, and 60X magnification. Total RNA was extracted from purified EVs using the miRNeasy Micro kit. RNA size distribution, concentration, and integrity were measured by the Agilent 6000 Pico Kit using Bioanalyzer. miRNA library was prepared using the NEBNext Multiplex Small RNA Library Prep kit. Samples were pooled and mixed with 135 and 146 bp custom-designed DNA ladder. Precise size selection was performed using agarose gel electrophoresis and quantified by NEB Next Library Quant kit using Quant Studio 5 real-time PCR system. Demultiplexed reads were pre-processed and trimmed to remove adapters fastp. FASTQ files were processed per ENCODE recommendations and aligned to human miRNA sequences (GRCh38/hg38) using the STAR aligner. DESeq2 was utilized for normalization due to nucleotide lengths.


Results, Conclusions, and Discussions:: cFCM indicated 55% exosomes from serum, 31% from plasma, and 30% from urine to be CD63+. Similar analysis showed 22% exosomes from serum, 41% from plasma, and 34% from urine to be CD81+. iFCM revealed high CD63+ expressions with 3.26 x 10⁷ EVs/mL for serum, 5.08 x 10⁶ EVs/mL for plasma, and 1.3 x 10⁷EVs/mL for urine. CD81+ expressions also revealed high yield with 1.32 x 10⁸ EVs/mL, 2.05 x 10⁶ EVs/mL, and 4.02 x 10⁶ EVs/mL for serum, plasma, and urine, respectively. Percentage of sEVs positive for each marker were comparable to expressions for sEVs purchased from ATCC Inc. Following miRNA sequencing, hsa-miR-6236, hsa-miR-148a, and hsa-let7b were found to be most highly enriched across samples. PCA, with 54% coverage, indicated urine sEVs segregated to +PC1, while serum and plasma sEVs intermixed in a -PC1 cluster. Plasma samples were furthermore found to cluster on -PC2, while most coming from serum remained on +PC2.
Analysis of sEVs isolated using the iDEP device was found comparable to those isolated using conventional techniques, including differential ultracentrifugation (DU) and size-exclusion chromatography (SEC). cFCM analysis has previously indicated 38.1% exosomes positive for both CD63 and CD81 when using DU for isolation from Broncho Alveolar Lavage fluid samples4. iFCM analyses have reported comparable CD63+ expressions (~1 x 10⁷ EVs/mL for DU and 3 X 10⁷ EVs/mL for SEC) and CD81+ (1.5 x 10⁶ EVs/mL for DU) from plasma and culture media, respectively^5,6. Additionally, miRNAs known to be highly expressed in cancers were observed to be enriched across sample types⁷. This affirms our isolation methodology as a viable alternative to those currently established.
The capacity and utility of a label-free iDEP device in isolating sEVs from serum, plasma, and urine was demonstrated by performing NTA and multiparametric characterization using cFCM, iFCM, and miRNA sequencing. The comprehensive characterization verified that sEVs were successfully isolated from biofluids with minimal impact to vesicles’ integrity. The iDEP device hence has potential to be further evolved as a simple yet powerful liquid biopsy platform for rapid isolation of sEVs based on their size and dielectric properties in clinical settings.


Acknowledgements (Optional): :

References (Optional): :
1. 1. Shi et al., Sci Rep, 2018




2. 2. Shi et al., Lab Chip, 2019




3. 3. Shi et al., Plos One, 2019




4. 4. Hough et al., Methods, 2020




5. 5. Mastoridis et al., Front Immunol, 2018




6. 6. Gorgens et al., J Extracell Vesicles, 2019




7. 7. Bertoli et al., Theranostics, 2015