Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Abstract

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Introduction

Extracellular vesicles (EVs) are cell-derived membrane-bound particles comprising two main types: exosomes (30-200 nm) and microvesicles (200-1000 nm)1. Exosomes form through inward budding of the endosomal membrane within a multivesicular body (MVB), releasing intraluminal vesicles (ILVs) into the extracellular space upon fusion with the plasma membrane1. In contrast, microvesicles are generated by outward budding and fission of the cell membrane2. EVs play a crucial role in intercellular communication by transporting proteins, nucleic acids, lipids, and metabolites, reflecting the physiological ....

Protocol

Sample collection was approved by the Latvian University Life and Medical Science Research Ethics Committee (decision N0-71-35/54)

NOTE: The materials used in this study are included in the Table of Materials file.

1. Three-dimensional (3D) printed mold fabrication

  1. Design a serpentine-shaped double negative mold in CAD software with the following dimensions for the top channel: height of 0.5 mm, width of 1 mm, and length.......

Representative Results

We fabricated a microfluidic device using a 3D printed double negative mold (Figure 1) via soft-lithography (Figure 2A) for high throughput EV separation based on the bifurcated A4F principle (Figure 2B,C). The setup requires a pump and a flow-through station, as can be seen in Figure 3, for the isolation of EVs in an automated manner. Firstly, to evaluate the proof of concept of the de.......

Discussion

The presented microfluidic device offers a promising method for the isolation and extraction of EVs from biological fluids, addressing some of the critical limitations of existing gold standard methods such as UC and SEC12. UC and SEC are known to be labor-intensive, time-consuming, and suffer from low yield, making them less suitable for high-throughput applications where large quantities of EVs are needed21,22. In contrast, the microflui.......

Acknowledgements

We thank all the donors who participated in this study, the staff of the Latvian Genome Database for providing the samples. The Institute of Solid-State Physics, University of Latvia as the Center of Excellence has received funding from the European Union's Horizon 2020 Framework Programme H2020-WIDESPREAD-01-2016-2017-TeamongPhase2 under grant agreement No. 739508, project CAMART2. This work was supported by The Latvian Council of Science Project No. lzp-2019/1-0142 and Project No: lzp-2022/1-0373.

....

Materials

NameCompanyCatalog NumberComments
0.1 µm carboxylate FluoSpheresInvitrogen#F8803Stock concentration: 3.6 x 1013 beads/mL (LOT dependent)
0.5 mL microcentrifuge tubesStarstedt72.704
1 mL Luer cone syringe single use without needleRAYSTUB1ML
1.0 µm polystyrene FluoSpheresInvitrogen#F13083Stock concentration: 1 x 1010 beads/mL (LOT dependent)
10 mL Serological pipettesSarstedt86.1254.001
15 mL (100k) Amicon Ultra centrifugal filtersMerck MilliporeUFC910024
2.0 mL Protein LoBind tubesEppendorf30108132
20 mL syringesBD PlastikPak10569215
250 µm ID polyether ether ketone tubingDarwin MicrofluidicsCIL-1581
3 kDa MWCO centrifugal filter unitsMerck Millipore,UFC200324
5 mL Medical Syringe without NeedleAnhui Hongyu Wuzhou Medical159646
50 mL conical tubesSarstedt62.547.254
70 Ti fixed angle ultracentrifuge rotorBeckman Coulter337922
800 µm ID polytetrafluoroethylene tubingDarwin MicrofluidicsLVF-KTU-15
96 well microplate, f-bottom, med. bindingGreiner Bio-One655001ELISA plate
B-27 Supplement (50x), serum freeThermo Fisher Scientific17504044
Bovine serum albuminSigmaAldrichA7906-100G
COC Topas microscopy slide platformMicrofluidic Chipshop10000002
COC Topas microscopy slide platform 2 x 16 Mini Luer Microfluidic Chipshop10000387
Elveflow OB1 pressure controllerElvesys Group
Luer connectorsDarwin Microfluidics CS-10000095
Mask aligner Suss MA/BA6SUSS MicroTec Group
Mixer Thinky ARE-250Thinky Corporation
NanoSight NS300Malvern PanalyticalNS300nanoparticle analyzer 
Optical microscope Nikon Eclipse LV150NNikon Metrology NV
OSTE 322 Crystal ClearMercene Labs
PBS TABLETS.Ca/Mg free. Fisher Bioreagents. 100 gFisher ScientificBP2944-100
PC membrane (50 nm pore diameter, 11.8% density)it4ip S.A., Louvain-La Neuve, Belgium
Petri dishes, sterileSarstedt82.1472.001
Plasma Asher GIGAbatch 360 MPVA TePla America, LLC
qEVoriginal/35 nm columnIzonSP5SEC column
QSIL 216 Silicone Elastomer KitPP&S
Resin Tough BlackZortrax
SW40 Ti swing ultracentrifuge rotorBeckman Coulter331301
Syringe pumpDK InfusetekISPLab002
T175 suspension flaskSarstedt83.3912.502
TIM4-Fc proteinAdipogen LifeSciencesAG-40B-0180B-3010
TMB (3,3',5,5'-tetramethylbenzidine)SigmaAldrichT0440-100MLHorseradish peroxidase substrate
Tween20SigmaAldrichP1379-100ML
Ultracentrifuge Optima L100XPBeckman Coulter
Ultrasonic cleaning unit P 60 HElma Schmidbauer GmbH
Universal Microplate SpectrophotometerBio-Tek instruments71777-1
Urine collection cup, 150mL, sterileAPTACA2120_SG
Whatman Anotop 25 Syringe FilterSigmaAldrich68092002
Zetasizer Nano ZSMalvern Panalyticaldynamic light scattering (DLS) system 
Zortrax InkspireZortrax

References

  1. Colombo, M., et al. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 30, 255-289 (2014).
  2. Tricarico, C., et al. Biology and biogenesis ....

Explore More Articles

Extracellular VesiclesEV IsolationAsymmetrical Flow Field flow FractionationMicrofluidic DeviceCOC OSTEContinuous FlowLarge volume SamplesBiomedical ApplicationsDiagnosticsDrug DeliveryRegenerative Medicine

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved