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.

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

<ol>
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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.

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

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

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.

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. ...

single-step-isolation-extracellular-vesicles-from-large-volume

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JoVE Journal

Bioengineering

603 Views.  Latvian Biomedical Research and Study Centre. 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. 

Video: Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.