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 size1-3. 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.
Results: 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 107 EVs/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.
Discussion: 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, respectively5,6. 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.
Conclusion: The 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 liquid biopsy platform for rapid isolation of sEVs based on their size and dielectric properties in clinical settings.

Leyla Esfandiari

Associate Professor of Biomedical Engineering

University of Cincinnati

Dr. Leyla Esfandiari completed her doctoral degree in Bioengineering from the University of California Los Angeles in 2014. She is currently an Associate Professor of Biomedical Engineering at the University of Cincinnati. She holds appointments in both Electrical Engineering and Environmental and Public Health Sciences at College of Medicine. Also, she is a member of the Cincinnati Cancer Center and the Center for Stem Cell and Organoid Medicine (CuSTOM) at the Cincinnati Children's Hospital. Her research is mainly focused on developing miniaturized devices, such as sensors and actuators, that can be applied in liquid biopsy and regenerative medicine. Her research has been supported by the National Institute of Health (NINDS, NIGMS, NCI), the National Science Foundation (ECCS, BMAT), and the Department of Defense (CDMRP). Dr. Esfandiari has won many awards, such as the National Institute of Health, Maximizing Investigator Research Award (MIRA) in 2023, the National Science Foundation CAREER Award in 2021, and the Engineering and Applied Sciences Distinguished Research Award in 2020 and 2023.

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Multiparametric analysis of small extracellular vesicles purified by a rapid and label-free lab on a chip device
Open to view video.  |   Closed captions available
Open to view video.  |   Closed captions available 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 size1-3. 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. Results: 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 107 EVs/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. Discussion: 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, respectively5,6. 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. Conclusion: The 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 liquid biopsy platform for rapid isolation of sEVs based on their size and dielectric properties in clinical settings.
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