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* These authors contributed equally
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.
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.
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 ....
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
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.......
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.......
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.
....Name | Company | Catalog Number | Comments |
0.1 µm carboxylate FluoSpheres | Invitrogen | #F8803 | Stock concentration: 3.6 x 1013 beads/mL (LOT dependent) |
0.5 mL microcentrifuge tubes | Starstedt | 72.704 | |
1 mL Luer cone syringe single use without needle | RAYS | TUB1ML | |
1.0 µm polystyrene FluoSpheres | Invitrogen | #F13083 | Stock concentration: 1 x 1010 beads/mL (LOT dependent) |
10 mL Serological pipettes | Sarstedt | 86.1254.001 | |
15 mL (100k) Amicon Ultra centrifugal filters | Merck Millipore | UFC910024 | |
2.0 mL Protein LoBind tubes | Eppendorf | 30108132 | |
20 mL syringes | BD PlastikPak | 10569215 | |
250 µm ID polyether ether ketone tubing | Darwin Microfluidics | CIL-1581 | |
3 kDa MWCO centrifugal filter units | Merck Millipore, | UFC200324 | |
5 mL Medical Syringe without Needle | Anhui Hongyu Wuzhou Medical | 159646 | |
50 mL conical tubes | Sarstedt | 62.547.254 | |
70 Ti fixed angle ultracentrifuge rotor | Beckman Coulter | 337922 | |
800 µm ID polytetrafluoroethylene tubing | Darwin Microfluidics | LVF-KTU-15 | |
96 well microplate, f-bottom, med. binding | Greiner Bio-One | 655001 | ELISA plate |
B-27 Supplement (50x), serum free | Thermo Fisher Scientific | 17504044 | |
Bovine serum albumin | SigmaAldrich | A7906-100G | |
COC Topas microscopy slide platform | Microfluidic Chipshop | 10000002 | |
COC Topas microscopy slide platform 2 x 16 Mini Luer | Microfluidic Chipshop | 10000387 | |
Elveflow OB1 pressure controller | Elvesys Group | ||
Luer connectors | Darwin Microfluidics | Â CS-10000095 | |
Mask aligner Suss MA/BA6 | SUSS MicroTec Group | ||
Mixer Thinky ARE-250 | Thinky Corporation | ||
NanoSight NS300 | Malvern Panalytical | NS300 | nanoparticle analyzer |
Optical microscope Nikon Eclipse LV150N | Nikon Metrology NV | ||
OSTE 322 Crystal Clear | Mercene Labs | ||
PBS TABLETS.Ca/Mg free. Fisher Bioreagents. 100 g | Fisher Scientific | BP2944-100 | |
PC membrane (50 nm pore diameter, 11.8% density) | it4ip S.A., Louvain-La Neuve, Belgium | ||
Petri dishes, sterile | Sarstedt | 82.1472.001 | |
Plasma Asher GIGAbatch 360 M | PVA TePla America, LLC | ||
qEVoriginal/35 nm column | Izon | SP5 | SEC column |
QSIL 216 Silicone Elastomer Kit | PP&S | ||
Resin Tough Black | Zortrax | ||
SW40 Ti swing ultracentrifuge rotor | Beckman Coulter | 331301 | |
Syringe pump | DK Infusetek | ISPLab002 | |
T175 suspension flask | Sarstedt | 83.3912.502 | |
TIM4-Fc protein | Adipogen LifeSciences | AG-40B-0180B-3010 | |
TMB (3,3',5,5'-tetramethylbenzidine) | SigmaAldrich | T0440-100ML | Horseradish peroxidase substrate |
Tween20 | SigmaAldrich | P1379-100ML | |
Ultracentrifuge Optima L100XP | Beckman Coulter | ||
Ultrasonic cleaning unit P 60 H | Elma Schmidbauer GmbH | ||
Universal Microplate Spectrophotometer | Bio-Tek instruments | 71777-1 | |
Urine collection cup, 150mL, sterile | APTACA | 2120_SG | |
Whatman Anotop 25 Syringe Filter | SigmaAldrich | 68092002 | |
Zetasizer Nano ZS | Malvern Panalytical | dynamic light scattering (DLS) system | |
Zortrax Inkspire | Zortrax |
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