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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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 microfluidic device described can continuously process a substantial volume of up to 20 mL of biological fluid with minimal user input, making it a potential game-changer for industrial or clinical settings. One of the key advantages of this microfluidic device is its ability to standardize EV isolation and improve reproducibility compared to traditional methods that involve manual steps. By reducing the variability of EV isolation device manufacturing through mass manufacturing, the device&#39;s performance can be better controlled and consistent, which is vital for applications like EV-based therapeutics and diagnostics. Furthermore, the device&#39;s compatibility with large-volume manufacturing via reaction injection molding of suitable substrates further enhances its potential for scalability and broader adoption.
To ensure reproducibility and consistent performance, an extensive and well-defined protocol is necessary, both to produce the chip and for its usage in experiments. The chip is designed with industrialization in mind. However, during the device fabrication in the laboratory environment, attention must be paid to avoiding the formation of bubbles, ensuring precise and robust bonding, and conducting thorough testing at higher flow speeds than those expected in real experiments. This precaution is especially important for COC-OSTE devices, as they can be prone to leaking due to unsuccessful bonding, especially at higher flow rates. Additionally, since OSTE is a photosensitive material, the device fabrication should be performed in yellow light to avoid unnecessary exposure to UV. As this technology is still in its initial stages, researchers should experiment with different flow speeds to identify the optimal setting for their specific application since standardized protocols for this novel method are yet to be established, and liquid viscosity can clearly affect EV isolation efficacy based on our results. By following these guidelines and addressing the challenges of standardization, the potential of this microfluidic device for EV isolation can be fully realized across various research areas and applications.
The method of EV isolation using the microfluidic device allows for extensive modification and troubleshooting to optimize performance. To improve particle separation, researchers can explore longer channels, different membrane porosities and pore densities, and altered dimensions. If there are a lot of large particles in the small particle outlet, testing a higher buffer inlet speed can be beneficial, while if there are many small particles in the large particle outlet, experimenting with a longer channel or a membrane with larger pores is recommended. Addressing particle absorption can involve adjusting flow speeds, surface modifications, and OSTE composition or changing the UV exposure time. For devices experiencing instability and leakage, decreasing flow speeds and ensuring proper gluing are crucial steps. To minimize EV damage, researchers can reduce flow speeds, wash with PBS more thoroughly after device sterilization, and consider using smaller membrane pores or alternative mold design techniques and materials. Through these adjustments, the microfluidic device can be fine-tuned, unleashing its potential for diverse applications in EV research and related fields. Additionally, to optimize the device&#39;s performance, upstream sample characterization, including density and viscosity measurements, could be incorporated to adjust flow parameters. Moreover, particular emphasis must be placed in the nature of the sample, given the established non-sterility of biofluids23.&#160;Consequently, the sterilization of samples before their administration in clinical applications should be duly considered.
Despite these challenges, the microfluidic device holds immense potential in various research areas. Its ability to continuously isolate EVs from large volumes of highly heterogeneous samples opens the door for automation, facilitating high-throughput applications and enabling more in-depth analysis of EVs from diverse sources. Integrating this microfluidic module into different devices, such as hollow fiber cell bioreactor cartridges or high-resolution flow cytometers, could make EV applications more industry-friendly and drive innovation in fields like drug delivery, diagnostics, and cosmetics4. Overall, this microfluidic device represents a significant step forward in the field of EV research, offering an efficient and standardized method for EV isolation with promising applications in a wide range of research an industrial settings.

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><tbody><tr><td>0.1 &amp;micro;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 &amp;micro;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 &amp;micro;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 &amp;micro;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&amp;nbsp;</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>&amp;nbsp;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&amp;nbsp;</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&amp;amp;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&#039;,5,5&#039;-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&amp;nbsp;</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 devices, a mixture of polystyrene beads with diameters of 100 nm and 1000 nm was prepared to represent vesicles and fine cell debris, respectively10.&#160;Experiments were conducted with varying flow rates for the bead mix, both with and without bifurcating flow, to investigate the effect of linear velocity on separation efficiency. Across all experiments, the recovery of small beads remained consistent and above 90%10,showing the potential of the device to recover EVs.
Then, we assessed and compared the potential of the COC-OSTE device in isolating EVs from large volumes (&#62;1 mL) from complex biofluids with minimal pre-processing. As such, urine from 10 healthy donors (Figure 4) and cell media from two different prostate cell lines (Figure 5) were used as a template to simultaneously isolate EVs following three different procedures: ultracentrifugation (UC), size exclusion chromatography (SEC), and the A4F microfluidics-based OSTE-COC device. After isolation, the total number of particles and their size distribution were assessed using nanoparticle tracking analysis (NTA). On average, the OSTE-COC device showed better total particle recovery from biofluids compared to UC, but the statistical significance was only achieved when combining the particle numbers from both ports (Figure 4A). In order to compare the device performance with other systems, R &#38; L outlet together should be taken into consideration. As shown in Figure 4, L &#38; R outlet together performance on recovering EVs outperforms UC and SEC. Separately, the L-port was designed to capture the small EV fraction, while the R-port was designed to collect the bigger EV fraction with other molecules of similar size. Interestingly, the recovery using the R-PORT of the OSTE-COC device was slightly higher than SEC and UC alone (Figure 4A). CD63 expression showed a similar pattern (Figure 4C). This finding indicates that the OSTE-COC device was more effective in total EV recovery. Equivalent size distribution was found between the different methodologies, except for UC, which shows a bigger particle size distribution (Figure 4B).
Comparable results were observed in cell media cultures. In both scenarios, the total particle recovery from both device ports exhibited superior performance compared to SEC or UC methodologies (as depicted in Figure 5A,B). Notably, EVs derived from PC3 cells demonstrated a distinct size distribution, with greater homogeneity in the L-PORT distribution when contrasted with other experimental groups (Figure 5C,D). Furthermore, the analysis of CD63 expression confirmed the higher EV recovery rates achieved using the COC-OSTE device (as illustrated in Figure 5E,F). A summary comparing the isolation characteristics of the different methodologies examined in this study can be found in Table 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66019/66019fig01.jpg" /> 
Figure 1: 3DP serpentine-shaped double negative mold dimensions and isometric view. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66019/66019fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66019/66019fig02.jpg" /> 
Figure 2: COC-OSTE microfluidic device. (A) Scheme of the different main steps of fabricating the OSTE-COC device. (B) Device working principle. (C) Image of the finished device. Scale bar: 15 mm. This figure has been modified with permission from Priedols et al.10 and Bajo-Santos et al.20. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66019/66019fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66019/66019fig03.jpg" /> 
Figure 3: Experimental configuration of the device. The syringe pump is on the left, the OSTE-COC device is in the middle, and the recovery station is on the right. This figure has been modified with permission from Priedols et al.10 and Bajo-Santos et al.20. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66019/66019fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66019/66019fig04.jpg" /> 
Figure 4: Urinary EV size distribution and particle recovery from 10 donors using ultracentrifugation (UC), size-exclusion chromatography (SEC), and the COC-OSTE device. (A)&#160;Particle amount recovered from each isolation method by nanoparticle tracking analysis (NTA). Data represented as mean +/- standard deviation. Statistical significance denoted by * (p&#60;0.05). (B) Boxplots display the average particle size distribution among all urine samples assessed by NTA, with whiskers indicating the minimum and maximum values. P-values were derived from comparisons to UC, with **** indicating high statistical significance (p&#60;0.0001). (C) The median and range of the average CD63 amount, assessed using double-sandwich enzyme-linked immunosorbent assay (dsELISA), for each isolation method, were calculated across all samples. L-port: Left port; R-port: Right port. This figure has been modified with permission from Bajo-Santos et al.20. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66019/66019fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66019/66019fig05.jpg" /> 
Figure 5: Characterization of EVs isolated from PC3 and LNCaP cells using different methods. (A)&#160;Particle amount recovered from PC3 media using nanoparticle tracking analysis (NTA) represented by mean+/- standard deviation. (B) Particle amount recovered from LNCaP cell media using NTA represented as mean +/- standard deviation. (C)&#160;Median particle size distribution of EVs from PC3 cultures, along with the range, was determined by NTA. (D) Median size distribution of EVs isolated from LNCaP cultures with range. (E) Median and range of CD63 expression for each isolation method for PC3 cell line-derived EV, using double-sandwich enzyme-linked immunosorbent assay (dsELISA). (F) Median and range of LNCaP-derived EV CD63 expression by dsELISA for each isolation method. UC: Ultracentrifugation; SEC: Size-exclusion chromatography; L-port: Left port; R-port: Right port.&#160;This figure has been modified with permission from Bajo-Santos et al.20. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66019/66019fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>UC</td>
			<td>SEC</td>
			<td>COC-OSTE</td>
		</tr>
		<tr>
			<td>Processing time/sample</td>
			<td>++/+++</td>
			<td>+++</td>
			<td>+</td>
		</tr>
		<tr>
			<td>Throughput</td>
			<td>++</td>
			<td>+</td>
			<td>+++</td>
		</tr>
		<tr>
			<td>Cost/sample</td>
			<td>+</td>
			<td>+++</td>
			<td>++</td>
		</tr>
		<tr>
			<td>Purity</td>
			<td>+</td>
			<td>+++</td>
			<td>++</td>
		</tr>
		<tr>
			<td>Automatization</td>
			<td>++</td>
			<td>+</td>
			<td>+++</td>
		</tr>
		<tr>
			<td>EV Yield</td>
			<td>++</td>
			<td>++</td>
			<td>+++</td>
		</tr>
		<tr>
			<td>Size selection</td>
			<td>+</td>
			<td>++</td>
			<td>++</td>
		</tr>
	</tbody>
</table>
Table 1. Comparison of isolation characteristics of EVs with the three methods UC SEC, and the COC-OSTE device. UC: Ultracentrifugation; SEC: Size-exclusion chromatography. COC-OSTE: cyclic olefin copolymer-off-stoichiometry thiol-ene. +: low; ++: medium; +++: high. * Device dependent.

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

643 Views.  Latvian Biomedical Research and Study Centre. We zien het potentieel van extracellulaire blaasjes, maar erkennen ook de moeilijkheid om ze te isoleren, vooral van grote biovloeistoffen zoals bioreactormedia. In dit onderzoek hebben we getest of we asymmetrische flow veld-flow fractionering kunnen gebruiken om extracellulaire blaasjes te isoleren. Op dit moment is het onmogelijk om extracellulaire blaasjes rechtstreeks uit biovloeistoffen te isoleren in een continue stroom voor integratie in bioreactoren.In plaats daarvan moeten bi...

Video: Auteur in de schijnwerpers: Asymmetrische veldstroomfractionering voor bioreactorintegratie

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 ...

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

Biochemistry

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Immunology and Infection

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...

Biology

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

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

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