Bifurcated Asymmetric Field Flow Fractionation of Nanoparticles in PDMS-Free Microfluidic Devices for Applications in Label-Free Extracellular Vesicle Separation

Abstract

Extracellular vesicles are small membrane-bound structures that are released by cells and play important roles in intercellular communication garnering significant attention in scientific society recently due to their potential as diagnostic and therapeutic tools. However, separating EVs from large-volume samples remains a challenge due to their small size and low concentration. In this manuscript, we presented a novel method for separating polystyrene beads as control and extracellular vesicles from large sample volumes using bifurcated asymmetric field flow fractionation in PDMS-free microfluidic devices. Separation characteristics were evaluated using the control system of polystyrene bead mix, which offers up to 3.7X enrichment of EV-sized beads. Furthermore, in the EV-sample from bioreactor culture media, we observed a notable population distribution shift of extracellular vesicles. Herein presented novel PDMS-free microfluidic device fabrication protocol resulted in devices with reduced EV-loss compared to size-exclusion columns. This method represented an improvement over the current state of the art in terms of EV separation from large sample volumes through the use of novel field flow fractionation design.

Extracellular Vesicles Isolation from Large Volume Samples Using a Polydimethylsiloxane-Free Microfluidic Device

“…For the separation experiments, a flow rate of 250 µL/min for both the EV-containing sample and PBS were selected according to a previous volume flowrate parameter sweep using polystyrene beads (100 nm and 1000 nm) as a model system. This flow rate system provided the highest bead enrichment as compared to other flow rates [18].…”

“…In addition, both materials are characterized by low lipophilic molecule absorption, and the use of a combination of these materials provides a scalable manufacturing process via reaction injection molding [24]. Furthermore, the combination of OSTE and COC shows better reproducibility as compared to PDMS [18]. The pore size of the membrane was chosen to prohibit EV passage through the pores while allowing the passage of smaller molecules and proteins.…”

“…The microfluidic device was fabricated and tested as described in our previous research [18]. In short, the microfluidic chip was fabricated using the soft lithography process.…”

“…However, these methods allow manipulation only with small volumes of samples of just a few hundred microliters, or have complex fabrication and separation procedures [11]. A promising method for EV separation is A4F [18]. A4F-based devices utilize the force exerted orthogonally to the porous membrane in microfluidic channels, caused by field flow coupled with Poiseuille flow, that allows the efficient isolation of particles that have different lateral placements in the channel, caused by diffusion [19].…”

“…Therefore, devices based on this principle could be used for EV isolation from large volume samples, such as cell media from hollow fiber bioreactors to produce therapeutic EV, or urine for prostate cancer screening purposes. Proof of principle for bifurcated A4F devices has been shown to produce 3.7X enrichment of EV-sized polystyrene beads [18].…”