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    Biomanufacturing

    (C-90) A Spiral Inertial Microfluidic Device for Continuous Production of Extracellular Vesicle Products

    Thursday, October 12, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row C - Poster # 90
    Introduction:: Extracellular vesicles (EVs) are cell-derived nanoscale membranous particles released from virtually all mammalian cell types. Recently, EVs have emerged as potential next-generation therapeutic for a wide variety of diseases and injuries. One such application of EVs was shown with regard to COVID-19 as EVs isolated from lung cells were engineered to display a recombinant SARS-CoV-2 receptor-binding domain. These EVs showed efficacy as an inhalable COVID-19 vaccine that can be lyophilized and stored at room temperatures for months, avoiding cold chain issues with mRNA vaccines and exhibiting superior biodistribution to the lung compared to liposomes [1]. Previous work has also shown EVs to be effective wound repair agents [2]. Biomanufacturing EVs is a huge challenge however due to their inherent complexity, with scalable production and downstream separation being key bottlenecks. Current separation methods for EVs rely on non-scalable technologies such as ultracentrifugation as well as filtration methods prone to fouling that thus eliminate the potential for continuous biomanufacturing. The aim of this work is to overcome the limitations associated with EV biomanufacturing in order to enable their rapid realization as viable therapeutics and biodefense products. This will be accomplished by leveraging previous work on perfusion bioreactor-based scalable EV production [3] with development of an inertial microfluidic device for EV purification to create a continuous production process.

    Materials and Methods:: The inertial microfluidic devices constructed here are based on a previous work [4] and can be seen in Figure 1. These devices were designed to utilize and manipulate particle size and the passive hydrodynamic forces acting on particles flowing through a microfluidic channel of a specific geometry. The design of these devices were honed in using computational fluid dynamic simulations (COMSOL). The current designs of these device channels were a spiral geometry with a rectangular cross-section of 600 micrometers (µm) by 80 µm. Three different designs (V1, V2, and V3) were used with the main differences being the direction of flow and the outer dimensions of the spiral radius and the device itself (Figure 1). These devices were constructed out of polydimethylsiloxane (PDMS) elastomer (Sylgard 184, Dow Corning, Midland, MI, USA) which was cured in acrylic molds. These molds were machined using a Haas CM1 CNC (Haas Automation Inc, Onard, CA, USA). Once cured, PDMS devices were bound to glass slides via plasma treatment (PDC-32G, Harrick Plasma Inc, Ithaca, NY, USA). Testing of the devices was done using HEK293T cells (~12 to 13 µm in size) and green fluorescent particles that were 100 nanometers (nm) in size and meant to represent EVs. Testing for separation efficiency was performed on all three devices.

    Results, Conclusions, and Discussions::

    As seen in Figure 2, device V1 (n=1) showed peak cell separation from 1.0 to 2.0 milliliters per minute (mL/min). Here, separation of the HEK293T cells reached 79.63% at 2.0 mL/min. For device V2 (n=1), cell separation peaked at 76.87% at 2.5 mL/min. Cell separation efficiency was also 76.67% at 0.5 mL/min but dipped between 1.0 and 2.0 mL/min (Figure 3). For device V3 (n=3), cell separation was highest from 1.0 to 2.5 mL/min with a peak of 75.13% at 1.0 mL/min (Figure 4). The amount of the 100 nm particles still present in the inner channel outlet was no higher than 51.40% and no lower than 45.54% for all the devices tested at all the flow rates. 




    The devices utilized in this work take advantage of the passive hydrodynamic forces acting on particles flowing through a microfluidic channel of a specific geometry. Within a microchannel, neutrally-buoyant particles will experience a net inertial lift force. Adding curvilinearity to the microchannel will result in two counter rotating vortices (Dean vortices), which apply a drag force. Separation is thus based on the force balance between the lift and drag forces and the size of the particle, all determining the particle’s equilibrium position within the channel [4]. In theory, the larger HEK293T cells will be driven to the inner wall while the smaller EVs would be dominated by the Dean vortices and driven to the outer wall. These results show promising cell separation, with the maximum separation of 79.63% occurring in device V1 at 2.5 mL/min. EV isolation (100 nm particles) however is around 50%, with the amount of 100 nm particles still present in the inner channel ranging from 45.54% to 51.40%. While the cell separation is encouraging, EV isolation will need to be improved in order to increase the throughput and amount of viable EVs for therapeutic production. Modifications to the channel’s geometry can help to improve the amount of EVs that are truly isolated. In particular, trapezoidal geometries can improve the Dean vortices and drive more flow to the outer wall.



    Acknowledgements (Optional): :

    The authors would like to acknowledge the Food and Drug Administration's (FDA) Office of Counterterrorism and Emerging Threats (OCET) for providing funding for this work.



    References (Optional): :

    [1] Wang Z, Popowski KD, Zhu D, de Juan Abad BL, Wang X, Liu M, Lutz H, De Naeyer N, DeMarco CT, Denny TN, Dinh PC, Li Z, Cheng K. Exosomes decorated with a recombinant SARS-CoV-2 receptor-binding domain as an inhalable COVID-19 vaccine. Nat Biomed Eng. 2022;6(7):791-805. Epub 20220704. doi: 10.1038/s41551-022-00902-5. PubMed PMID: 35788687.




    [2] Born LJ, Chang KH, Shoureshi P, Lay F, Bengali S, Hsu ATW, Abadchi SN, Harmon JW, Jay SM. HOTAIR-Loaded Mesenchymal Stem/Stromal Cell Extracellular Vesicles Enhance Angiogenesis and Wound Healing. Adv Healthc Mater. 2021:e2002070. Epub 2021/04/20. doi: 10.1002/adhm.202002070. PubMed PMID: 33870645.




    [3] Patel DB, Luthers CR, Lerman MJ, Fisher JP, Jay SM. Enhanced extracellular vesicle production and ethanol-mediated vascularization bioactivity via a 3D-printed scaffold-perfusion bioreactor system. Acta Biomater. 2019;95:236-44. Epub 2018/11/25. doi: 10.1016/j.actbio.2018.11.024. PubMed PMID: 30471476; PMCID: PMC6531369.



    [4] Kwon T, Prentice H, Oliveira, JD, Madziva N, Warkiani ME, Hamel JFP, Han J. Microfluidic cell retention device for perfusion of mammalian suspension culture. Scientific Reports. 2017; 7(1). doi: 10.1038/s41598-017-06949-8