Poster Z5 - SELECTIVE CAPTURE AND ISOLATION OF EXTRACELLULAR VESICLES VIA DIAMAGNETIC MICROFLUIDIC SORTING

Introduction: EVs constitute lipid bilayer vesicles secreted by cells, ranging from 30 nm to 10 µm in size. They encapsulate proteins, nucleic acids, lipids, and metabolites and play critical roles in cellular communication, including tumor cell migration, proliferation, and immunosuppression within the tumor microenvironment. However, the heterogeneity of EVs poses challenges for their isolation, and therefore characterization. Despite the various methods have been developed (e.g., ultracentrifugation, polymer precipitation, size-exclusion chromatography, and immuno-magnetic capture) limitations in terms of handling the small sample volumes and selective isolation yet persist. Here, we introduce a technique for selective isolation of EV subpopulations based on common tetraspanins on EV surface: CD9, CD63, and CD81. Our system integrates microfluidics and polymer beads of two different densities to capture EV subpopulations. Those beads that carry EV subpopulations are collected at different outlets (i.e., top and bottom) in our platform. This innovative approach enables rapid and simultaneous isolation of EVs from small volume samples (sub-milliliter), overcoming the limitations of traditional methods.

Materials and

Methods: We usepolystyrene (PS) and Polymethyl methacrylate (PMMA) beads, with densities of 1.05 and 1.18 g/mL respectively, coated with either CD9, CD63, or CD81 antibodies to capture different EV subpopulations (Fig 1A). Then, the beads are mixed with 0.2 µm syringe-filtered human plasma (Fig 1B). After washing (Fig 1C), EV-carrying beads are mixed with an FDA-approved paramagnetic agent (Gadavist) and loaded into the microfluidic platform where the bead mixture is fed into the system via syringe pumps using 50 µL and 10 µL/min withdrawal rates for top and bottom channels, respectively (Fig 1D). Our portable design consists of two magnets facing the same poles each other (2x5x50 mm) and a glass fluidic channel (1x1x50 mm) attached to two outlets and one inlet channel. Since the beads are inherently diamagnetic, they can be collected at different outlets of our platform based on their density profile via diamagnetophoresis. Lastly, EVs on collected beads (Fig 1E) are eluted (Fig 1F) and characterized using Interferometric Scattering (iSCAT) Microscopy, Flow Cytometry (FC), and Transmission Electron Microscopy (TEM) for size and particle concentration, tetraspanin expression, and morphology analysis, respectively.

Results, Conclusions, and Discussions: We presented a tool that leverages the difference in diamagnetophoresis of polymer beads, allowing efficient EV isolation. iSCAT microscopy results confirmed that the collected EVs were in small EV size range and TEM analyses confirmed the preservation of EV structural integrity post-isolation (Fig 2 and B). The method yielded a concentration of 9.83 x 106 ± 4.77 x 106 particle/mL and 9.85 x 106 ± 3 x 106 particle/mL for top and bottom channels, respectively. The mean size of the eluted particles were 105.3 ± 6.2 nm and 105.3 ± 6.6 nm for top and bottom channels, respectively. TEM images also showed this method produced clean, pure, and uniform EVs from human plasma. FC analysis confirmed the tetraspanin signals on eluted EVs.
We believe this technology could offer a portable, cost-effective, and straightforward assay scheme to be used in uncovering the cargo and function of EV subpopulations associated with various diseases, including cancer and infectious diseases as well as in discovering disease biomarkers in small volume samples. Since the method is extremely versatile, it has the potential to sort any biological molecules rapidly and accurately with known surface markers.

Acknowledgements (Optional): Funding was provided through the Career Award at the Scientific Interface (CASI) from the Burroughs Wellcome Foundation (BWF), McCormick and Gabilan Faculty Award from Stanford University, and the Donald E. and Delia B. Baxter Foundation.