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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

Sample collection was approved by the Latvian University Life and Medical Science Research Ethics Committee (decision N0-71-35/54)

NOTE: The materials used in this study are included in the Table of Materials file.

1. Three-dimensional (3D) printed mold fabrication

1. Design a serpentine-shaped double negative mold in CAD software with the following dimensions for the top channel: height of 0.5 mm, width of 1 mm, and length of 210 mm. For the bottom channel, use the same design with a width of 1.5 mm, height of 0.6 mm, and length of 210 mm. Dimensions such as mold width,....

Results

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.......

Discussion

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 needed^21,22. In contrast, the microflui.......

Materials

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