urine testing for extracellular vesicle isolation using a bilayer microfluidic device

Mikrofluidisk enhed til kontinuerlig ekstracellulær vesikelisolering fra human urin

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.

Forfatter Spotlight: Asymmetrisk feltflowfraktionering til bioreaktorintegration

Enkelttrinsisolering af ekstracellulære vesikler fra prøver med stort volumen med en forgrenet A4F mikrofluidisk enhed

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.

Urintest for ekstracellulær vesikelisolering ved hjælp af en dobbeltlags mikrofluidisk enhed

Til at begynde med skal du placere et oxideret COC-dias på en PDMS-form til enhedens øverste kanal. Anbring samlingen i en form og læg jig. Stram derefter jiggen med en momentnøgle indstillet til 0,3 newtonmeter.Tilslut PTFE-røret til formens indløb. Fyld enheden med 1.000 millibar tryk, der opretholdes af Piezo elektrisk pumpe placeret i tryksystemet. Placer jiggen med glasgliden op i maskejusteringen, og udsæt den for 850 millijoule dosis med et ND33-filter.Bring nu den strukturerede OSTE i kontakt med membranen med 11,8% dårlig densitet, hvilket sikrer, at kanalerne er fuldt dækket af membranen. Placer et rent glasglas oven på membranen. Påfør et tryk på 1,6 kilopascal fra toppen for at sikre jævn binding.Placer derefter monteringsmembransiden opad på en kogeplade ved 60 grader Celsius. Juster derefter strukturen OSTE-laget med den øverste kanalsamling. Sørg for, at det øverste og nederste lag er justeret, og at OSTE-bundlaget er i kontakt med membranen.Brug et mikroskop til visuelt at evaluere enheden for eventuelle defekter. Når du er færdig, skal du fylde to fem milliliter sprøjter med to milliliter hydrogenperoxid. Sæt sprøjterne på en sprøjtepumpe.Tilslut derefter 14 centimeter lang PTFE-slange til sprøjterne med sprøjtenålene. Tilslut slangen til enhedens indløb med luer-stik. Tilslut 200 centimeter lang PEEK-slange til enhedens stikkontakter.Tilslut derefter den anden ende af PEEK-slangen til en affaldsbeholder. Tryk på Indstillinger på instrumentskærmen, og vælg derefter Systemsæt efterfulgt af Sprøjte. Klik derefter på tilbage-knappen og vælg parametre for at indtaste en strømningshastighed på 500 mikroliter pr. Minut.Opsaml strømmen igennem i en affaldsbeholder. Fyld derefter to nye fem milliliter sprøjter med to ml ethanol. Udskift de brugte sprøjter med de ethanolfyldte sprøjter, og start derefter pumpen.På samme måde fyldes sprøjter med fem ml 20 nanometer filtreret PBS. Tilslut dem til pumpen, og skyl enheden ud med fire ml PBS-opløsning. Tag slangen ud af enheden.Sæt indløb og stikkontakter med luer-stik. Den næste dag skylles den PBS-fyldte enhed med fire ml filtreret PBS. Opsaml den sidste milliliter af strømmen fra begge udløb i et to-milliliter protein lavbindingsrør.Opbevares ved fire grader Celsius som et emne for partikler, der indføres af enheden. Udskift de PBS-fyldte sprøjter med luftfyldte. Sæt derefter enheden på plads med fire milliliter luft, indtil al væsken er kommet ud af enheden og slangen.For at teste urinprøver skal du først samle 100 ml morgenurin fra hver donor i sterile beholdere. Hver prøve adskilles i to 50 ml rør. Centrifuger rørene ved 2.000 G i 15 minutter ved stuetemperatur.Derefter samles supernatanten. Supernatanten centrifugeres ved 10.000 G i 30 minutter ved stuetemperatur for at fjerne eventuelle partikler. Supernatanten samles i nye glas.Fyld en 20 ml sprøjte med den forberedte urinprøve, og tilslut den derefter til enhedens prøveindgang. Fyld derefter en anden 20 ml sprøjte med 20 ml filtreret DEPC-behandlet PBS, og tilslut den til enhedens bufferindgang. Overfør strømmen til 100.000 molekylvægtsafskæringskoncentrationsrør separat, og centrifuger rørene for at koncentrere 100 mikroliter hver.Overfør derefter koncentraterne til 0,5 ml mikrocentrifugerør. Vortex koncentraterne i 30 sekunder, drej derefter i 10 sekunder i en centrifuge. Overfør 25 mikroliter af hver prøve til nye mærkede rør.Frys dem ved 80 grader Celsius til langtidsopbevaring. OSTE-COC-enheden viste bedre total partikelgenvinding fra biofluider i forhold til ultracentrifugering. Genvindingen ved hjælp af R-porten på OSTE-COC-enheden var lidt højere end størrelseseksklusionskromatografi og ultracentrifugering alene.En ækvivalent størrelsesfordeling blev fundet mellem de forskellige metoder med undtagelse af ultracentrifugering, som viste en større partikelstørrelsesfordeling. Den samlede partikelgenvinding fra begge enhedsporte udviste overlegen ydeevne.

urine-testing-for-extracellular-vesicle-isolation-using-bilayer

Research

JoVE Journal

Bioengineering

Urine Testing for Extracellular Vesicle Isolation Using a Bilayer Microfluidic Device

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.

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. ...