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. ...

University of Latvia

Cellbox labs LTD, Atari

Current in vitro models have significant limitations for new respiratory disease research and rapid drug repurposing. Lung on a chip (LOAC) technology offers a potential solution to these problems. However, these devices typically are fabricated from polydimethylsiloxane (PDMS), which has small hydrophobic molecule absorption, which hinders the application of this technology in drug repurposing for respiratory diseases. Off-stoichiometry thiol-ene (OSTE) is a promising alternative material class to PDMS. Therefore, this study aimed to test OSTE as an alternative material for LOAC prototype development and compare it to PDMS. We tested OSTE material for light transmission, small molecule absorption, inhibition of enzymatic reactions, membrane particle, and fluorescent dye absorption. Next, we microfabricated LOAC devices from PDMS and OSTE, functionalized with human umbilical vein endothelial cell (HUVEC) and A549 cell lines, and analyzed them with immunofluorescence. We demonstrated that compared to PDMS, OSTE has similar absorption of membrane particles and effect on enzymatic reactions, significantly lower small molecule absorption, and lower light transmission. Consequently, the immunofluorescence of OSTE LOAC was significantly impaired by OSTE optical properties. In conclusion, OSTE is a promising material for LOAC, but optical issues should be addressed in future LOAC prototypes to benefit from the material properties.

Lung on a Chip Development from Off-Stoichiometry Thiol-Ene Polymer.

The possibility to artificially adjust and fine-tune gene expression is one of the key milestones in bioengineering, synthetic biology, and advanced medicine. Since the effects of proteins or other transgene products depend on the dosage, controlled gene expression is required for any applications, where even slight fluctuations of the transgene product impact its function or other critical cell parameters. In this context, physical techniques demonstrate optimistic perspectives, and pulsed electric field technology is a potential candidate for a noninvasive, biophysical gene regulator, exploiting an easily adjustable pulse generating device. We exposed mammalian cells, transfected with a NF-κB pathway-controlled transcription system, to a range of microsecond-duration pulsed electric field parameters. To prevent toxicity, we used protocols that would generate relatively mild physical stimulation. The present study, for the first time, proves the principle that microsecond-duration pulsed electric fields can alter single-gene expression in plasmid context in mammalian cells without significant damage to cell integrity or viability. Gene expression might be upregulated or downregulated depending on the cell line and parameters applied. This noninvasive, ligand-, cofactor-, nanoparticle-free approach enables easily controlled direct electrostimulation of the construct carrying the gene of interest; the discovery may contribute towards the path of simplification of the complexity of physical systems in gene regulation and create further synergies between electronics, synthetic biology, and medicine.

Pulsed Electric Fields Alter Expression of NF-κB Promoter-Controlled Gene.

The majority of proposed exotic applications employing 3D topological insulators require high-quality materials with reduced dimensions. Catalyst-free, PVD-grown BiSe nanoribbons are particularly promising for these applications due to the extraordinarily high mobility of their surface Dirac states, and low bulk carrier densities. However, these materials are prone to the formation of surface accumulation layers; therefore, the implementation of surface encapsulation layers and the choice of appropriate dielectrics for building gate-tunable devices are important. In this work, all-around ZnO-encapsulated nanoribbons are investigated. Gate-dependent magnetotransport measurements show improved charge transport characteristics as reduced nanoribbon/substrate interface carrier densities compared to the values obtained for the as-grown nanoribbons on SiO substrates.

Magnetotransport Studies of Encapsulated Topological Insulator BiSe Nanoribbons.

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.

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

Extracellular vesicles (EV) have many attributes important for biomedicine; however, current EV isolation methods require long multi-step protocols that generally involve bulky equipment that cannot be easily translated to clinics. Our aim was to design a new cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) asymmetric flow field fractionation microfluidic device that could isolate EV from high-volume samples in a simple and efficient manner. We tested the device with large volumes of urine and conditioned cell media samples, and compared it with the two most commonly used EV isolation methods. Our device was able to separate particles by size and buoyancy, and the attained size distribution was significantly smaller than other methods. This would allow for targeting EV size fractions of interest in the future. However, the results were sample dependent, with some samples showing significant improvement over the current EV separation methods. We present a novel design for a COC-OSTE microfluidic device, based on bifurcating asymmetric flow field-flow fractionation (A4F) technology, which is able to isolate EV from large volume samples in a simple, continuous-flow manner. Its potential to be mass-manufactured increases the chances of implementing EV isolation in a clinical or industry-friendly setting, which requires high repeatability and throughput.

Gatis Mozolevskis

Institute of Solid-State Physics

University of Latvia

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

Gatis Mozolevskis

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Solid-State Physics

University of Latvia

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

Institute of Solid-State Physics