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&#39;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.

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
<ol>
	<li>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...

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

<ol>
	<li>Colombo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+secretion,+and+intercellular+interactions+of+exosomes+and+other+extracellular+vesicles.">Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.</a> Annu Rev Cell Dev Biol. 30, 255-289 (2014).</li><li>Tricarico, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+biogenesis+of+shed+microvesicles.">Biology and biogenesis of shed microvesicles.</a> Small GTPases. 8 (4), 220-232 (2017).</li><li>Y&#225;&#241;ez-M&#243;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+properties+of+extracellular+vesicles+and+their+physiological+functions.">Biological properties of extracellular vesicles and their physiological functions.</a> J Extracell Vesicles. 4 (1), 27066 (2015).</li><li>Th&#233;ry, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> J Extracell Vesicles. 7 (1), 1535750 (2018).</li><li>Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+as+an+emerging+drug+delivery+system+for+cancer+treatment:+Current+strategies+and+recent+advances.">Extracellular vesicles as an emerging drug delivery system for cancer treatment: Current strategies and recent advances.</a> Biomed Pharmacother. 153, 113480 (2022).</li><li>Royo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+separation+and+characterization+of+extracellular+vesicles:+Results+of+a+Worldwide+Survey+Performed+by+the+ISEV+Rigor+and+Standardization+Subcommittee.">Methods for separation and characterization of extracellular vesicles: Results of a Worldwide Survey Performed by the ISEV Rigor and Standardization Subcommittee.</a> Cells. 9 (9), 1955 (2020).</li><li>Sidhom, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+exosomal+isolation+methods:+Is+size+exclusion+chromatography+the+best+option.">A review of exosomal isolation methods: Is size exclusion chromatography the best option.</a> Int J Mol Sci. 21 (18), 6466 (2020).</li><li>Reshi, Q. U. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+extracellular+vesicles+(EVs)+using+benchtop+size+exclusion+chromatography+(SEC)+columns.">Isolation of extracellular vesicles (EVs) using benchtop size exclusion chromatography (SEC) columns.</a> Methods Mol Biol. 2273, 201-206 (2021).</li><li>Liangsupree, T., Multia, E., Riekkola, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+isolation+and+separation+techniques+for+extracellular+vesicles.">Modern isolation and separation techniques for extracellular vesicles.</a> J Chromatogr A. 1636, 461773 (2021).</li><li>Priedols, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bifurcated+asymmetric+field+flow+fractionation+of+nanoparticles+in+PDMS-free+microfluidic+devices+for+applications+in+label-free+extracellular+vesicle+separation.">Bifurcated asymmetric field flow fractionation of nanoparticles in PDMS-free microfluidic devices for applications in label-free extracellular vesicle separation.</a> Polymers. 15 (4), 789 (2023).</li><li>Zhang, H., Lyden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric-flow+field-flow+fractionation+technology+for+exomere+and+small+extracellular+vesicle+separation+and+characterization.">Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization.</a> Nat Protoc. 14 (4), 1027-1053 (2019).</li><li>Talebjedi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+microfluidics+for+extracellular+vesicle+isolation+and+characterization:+Potential+use+for+standardized+embryo+quality+assessment.">Exploiting microfluidics for extracellular vesicle isolation and characterization: Potential use for standardized embryo quality assessment.</a> Front Vet Sci. 7, 620809 (2020).</li><li>van Meer, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+absorption+by+PDMS+in+the+context+of+drug+response+bioassays.">Small molecule absorption by PDMS in the context of drug response bioassays.</a> Biochem Biophys Res Commun. 482 (2), 323-328 (2017).</li><li>Jia, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+extracellular+vesicles+isolation+and+separation:+Current+techniques,+pending+questions+and+clinical+applications.">Small extracellular vesicles isolation and separation: Current techniques, pending questions and clinical applications.</a> Theranostics. 12 (15), 6548-6575 (2022).</li><li>Bussooa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+oxygen+levels+within+thermoplastic+Organ-on-Chip+devices.">Real-time monitoring of oxygen levels within thermoplastic Organ-on-Chip devices.</a> Biosensors and Bioelectronics: X. 11, 100198 (2022).</li><li>Tang, L., Lee, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+facile+route+for+irreversible+bonding+of+plastic-PDMS+hybrid+microdevices+at+room+temperature.">A facile route for irreversible bonding of plastic-PDMS hybrid microdevices at room temperature.</a> Lab Chip. 10 (10), 1274-1280 (2010).</li><li>Borda, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformable+neural+interface+based+on+off-stoichiometry+thiol-ene-epoxy+thermosets.">Conformable neural interface based on off-stoichiometry thiol-ene-epoxy thermosets.</a> Biomaterials. 293, 121979 (2023).</li><li>Sandstr&#246;m, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+injection+molding+and+direct+covalent+bonding+of+OSTE++polymer+microfluidic+devices.">Reaction injection molding and direct covalent bonding of OSTE+ polymer microfluidic devices.</a> Journal of Micromechanics and Microengineering. 25 (7), 075002 (2015).</li><li>Sticker, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-ene+based+polymers+as+versatile+materials+for+microfluidic+devices+for+life+sciences+applications.">Thiol-ene based polymers as versatile materials for microfluidic devices for life sciences applications.</a> ACS Appl Mater Interfaces. 12 (9), 10080-10095 (2020).</li><li>Bajo-Santos, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+isolation+from+large+volume+samples+using+a+polydimethylsiloxane-free+microfluidic+device.">Extracellular vesicles isolation from large volume samples using a polydimethylsiloxane-free microfluidic device.</a> Int J Mol Sci. 24 (9), 7971 (2023).</li><li>Claridge, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+extracellular+vesicle+therapeutics:+Challenges,+considerations,+and+opportunities.">Development of extracellular vesicle therapeutics: Challenges, considerations, and opportunities.</a> Front Cell Dev Biol. 9, 734720 (2021).</li><li>Rezaie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+exosomes+application+in+clinical+trials:+Perspective,+questions,+and+challenges.">Review on exosomes application in clinical trials: Perspective, questions, and challenges.</a> Cell Commun Signal. 20 (1), 145 (2022).</li><li>Arag&#243;n, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+urinary+tract+microbiome+in+health+and+disease.">The urinary tract microbiome in health and disease.</a> Eur Urol Focus. 4 (1), 128-138 (2018).</li></ol>

A.A., G.M., and R.R. are founders, board members, and equity holders in Cellbox Labs, LLC

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...

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 state of the cell, including growth, angiogenesis, metastasis, proliferation, and therapy resistance3. As a result, they have emerged as promising biomarkers and therapeutic targets for diseases, including cancer, highlighting their potential in diagnostics and drug delivery systems4.
To fully utilize EVs in disease diagnostics and therapeutics, efficient isolation from various biofluids is crucial5.&#160;Common methods include ultracentrifugation (UC), density gradient centrifugation, size exclusion chromatography (SEC), filtration, and immunoisolation6. UC is a widely used technique but may yield particles of similar density that are not EVs and can generate EV aggregates7. SEC has gained popularity due to its ability to provide higher purity samples by excluding particles based on size rather than density8. However, careful selection of the appropriate pore size for the SEC column and optimization of chromatography conditions are essential to minimize co-isolation of unwanted particles like chylomicrons and low-density lipoproteins8. Despite their effectiveness, both methods are time-consuming and challenging to automate, especially for larger volume samples like cell media or urine, limiting their scalability for industrial applications9.
In recent years, asymmetric field flow field fractionation (A4F) has evolved as a powerful separation technique for size and buoyancy-based micro- and nanometer-sized particle separation10. The operational principle of A4F relies on a microfluidic channel endowed with a porous membrane at its base, generating a force exerted towards the membrane called cross-flow10. When combined with Brownian motion and Poiseuille flow inherent to the system, cross-flow facilitates efficient particle separation due to varying particle position within the flow dynamics11. Despite the benefits, this method is limited to sample volumes within the microliter range12 and requires an additional focusing step, extending the duration of the process10.
Over the last decade, microfluidics has gained prominence as a tool for rapid, efficient, and clinically reliable EV separation13. However, most microfluidic methods designed for EV separation are optimized for small-volume, high-concentration EV samples or depend on complex separation procedures14. Furthermore, within the field of microfluidics, polydimethylsiloxane (PDMS) is recognized as the golden standard material owing to its optical transparency, biocompatibility, and ease of use15. Nevertheless, its known propensity to absorb small lipophilic molecules, including EVs, can be problematic for its application in the EV field13.
Cyclic olefin copolymer (COC) is a frequently used material in microfluidics due to biocompatibility, small absorption of molecules, and high chemical resistance15. However, the fabrication of COC devices often involves complex processes or specialized equipment16. Alternatively, off-stoichiometry thiol-ene (OSTE) is a promising alternative to PDMS due to decreased absorption of small molecules, superior chemical stability, ease of fabrication, and scalable fabrication process17,18. However, due to complex connections to tubing, devices can be prone to leaking19.
The aim of this study was to engineer and fabricate a microfluidic device combining OSTE and COC and bifurcated A4F principle for EV separation from large-volume samples such as urine or cell media.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 &#181;m carboxylate FluoSpheres</td>
			<td>Invitrogen</td>
			<td>#F8803</td>
			<td>Stock concentration: 3.6 x 1013 beads/mL (LOT dependent)</td>
		</tr>
		<tr>
			<td>0.5 mL microcentrifuge tubes</td>
			<td>Starstedt</td>
			<td>72.704</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Luer cone syringe single use without needle</td>
			<td>RAYS</td>
			<td>TUB1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 &#181;m polystyrene FluoSpheres</td>
			<td>Invitrogen</td>
			<td>#F13083</td>
			<td>Stock concentration: 1 x 1010 beads/mL (LOT dependent)</td>
		</tr>
		<tr>
			<td>10 mL Serological pipettes</td>
			<td>Sarstedt</td>
			<td>86.1254.001</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL (100k) Amicon Ultra centrifugal filters</td>
			<td>Merck Millipore</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL Protein LoBind tubes</td>
			<td>Eppendorf</td>
			<td>30108132</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL syringes</td>
			<td>BD PlastikPak</td>
			<td>10569215</td>
			<td></td>
		</tr>
		<tr>
			<td>250 &#181;m ID polyether ether ketone tubing</td>
			<td>Darwin Microfluidics</td>
			<td>CIL-1581</td>
			<td></td>
		</tr>
		<tr>
			<td>3 kDa MWCO centrifugal filter units</td>
			<td>Merck Millipore,</td>
			<td>UFC200324</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Medical Syringe without Needle</td>
			<td>Anhui Hongyu Wuzhou Medical</td>
			<td>159646</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>70 Ti fixed angle ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>337922</td>
			<td></td>
		</tr>
		<tr>
			<td>800 &#181;m ID polytetrafluoroethylene tubing</td>
			<td>Darwin Microfluidics</td>
			<td>LVF-KTU-15</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well microplate, f-bottom, med. binding</td>
			<td>Greiner Bio-One</td>
			<td>655001</td>
			<td>ELISA plate</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>SigmaAldrich</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>COC Topas microscopy slide platform</td>
			<td>Microfluidic Chipshop</td>
			<td>10000002</td>
			<td></td>
		</tr>
		<tr>
			<td>COC Topas microscopy slide platform 2 x 16 Mini Luer&#160;</td>
			<td>Microfluidic Chipshop</td>
			<td>10000387</td>
			<td></td>
		</tr>
		<tr>
			<td>Elveflow OB1 pressure controller</td>
			<td>Elvesys Group</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luer connectors</td>
			<td>Darwin Microfluidics</td>
			<td>&#160;CS-10000095</td>
			<td></td>
		</tr>
		<tr>
			<td>Mask aligner Suss MA/BA6</td>
			<td>SUSS MicroTec Group</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer Thinky ARE-250</td>
			<td>Thinky Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight NS300</td>
			<td>Malvern Panalytical</td>
			<td>NS300</td>
			<td>nanoparticle analyzer&#160;</td>
		</tr>
		<tr>
			<td>Optical microscope Nikon Eclipse LV150N</td>
			<td>Nikon Metrology NV</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OSTE 322 Crystal Clear</td>
			<td>Mercene Labs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS TABLETS.Ca/Mg free. Fisher Bioreagents. 100 g</td>
			<td>Fisher Scientific</td>
			<td>BP2944-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PC membrane (50 nm pore diameter, 11.8% density)</td>
			<td>it4ip S.A., Louvain-La Neuve, Belgium</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, sterile</td>
			<td>Sarstedt</td>
			<td>82.1472.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Asher GIGAbatch 360 M</td>
			<td>PVA TePla America, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>qEVoriginal/35 nm column</td>
			<td>Izon</td>
			<td>SP5</td>
			<td>SEC column</td>
		</tr>
		<tr>
			<td>QSIL 216 Silicone Elastomer Kit</td>
			<td>PP&#38;S</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Resin Tough Black</td>
			<td>Zortrax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SW40 Ti swing ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>331301</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>DK Infusetek</td>
			<td>ISPLab002</td>
			<td></td>
		</tr>
		<tr>
			<td>T175 suspension flask</td>
			<td>Sarstedt</td>
			<td>83.3912.502</td>
			<td></td>
		</tr>
		<tr>
			<td>TIM4-Fc protein</td>
			<td>Adipogen LifeSciences</td>
			<td>AG-40B-0180B-3010</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB (3,3&#39;,5,5&#39;-tetramethylbenzidine)</td>
			<td>SigmaAldrich</td>
			<td>T0440-100ML</td>
			<td>Horseradish peroxidase substrate</td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>SigmaAldrich</td>
			<td>P1379-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge Optima L100XP</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaning unit P 60 H</td>
			<td>Elma Schmidbauer GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Microplate Spectrophotometer</td>
			<td>Bio-Tek instruments</td>
			<td>71777-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Urine collection cup, 150mL, sterile</td>
			<td>APTACA</td>
			<td>2120_SG</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Anotop 25 Syringe Filter</td>
			<td>SigmaAldrich</td>
			<td>68092002</td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer Nano ZS</td>
			<td>Malvern Panalytical</td>
			<td></td>
			<td>dynamic light scattering (DLS) system&#160;</td>
		</tr>
		<tr>
			<td>Zortrax Inkspire</td>
			<td>Zortrax</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single step isolation of extracellular vesicles from large-volume samples with a bifurcated a4f microfluidic device

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.

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

We see the potential of extracellular vesicles, but also recognize the difficulty in their isolation, especially from large sample biofluids such as bioreactor media. In this research we tested if we can use asymmetrical flow field-flow fractionation to isolate extracellular vesicles. As of now, isolating extracellular vesicles directly from biofluids in a continuous flow for integration into bioreactors is impossible.Instead, biofluids must be first collected and processed, adding variability and labor. Our device designed for extracellular vesicle isolation from large volume biofluids enables integration with bioreactors for continuous flow and automation. We found liquid viscosity might impact extracellular vesicles isolation using asymmetric field flow fractionation.We're exploring if adapting biofluid and buffer flows to viscosity can enhance isolation.

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...

Watch this Scientific Journal Video about Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration at JoVE.com

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. ...

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Research

JoVE Journal

Bioengineering

432 Views.  Latvian Biomedical Research and Study Centre. We see the potential of extracellular vesicles, but also recognize the difficulty in their isolation, especially from large sample biofluids such as bioreactor media. In this research we tested if we can use asymmetrical flow field-flow fractionation to isolate extracellular vesicles. As of now, isolating extracellular vesicles directly from biofluids in a continuous flow for integration into bioreactors is impossible.Instead, biofluids must be first collected and processed, adding var...