2025 AIChE Annual Meeting

Establishing Parallel Flow for Dynamic Nanopore Microfluidic Transfection Via Electroporation (DyNaMiTE)

Nanopore electroporation (NanoEP), an efficient and gentle nonviral transfection method, allows for delivery of macromolecules such as proteins, DNA, and mRNA into cells. In NanoEP, an applied voltage is localized within the nanopores of a polycarbonate track-etched (PCTE) membrane, temporarily forming pores along the membranes of adhered cells and driving a “cargo cocktail” of macromolecules into the cells through electrophoretic force. However, NanoEP currently exists in a stationary environment, limiting delivery to just one cargo cocktail. We propose to integrate NanoEP with active flow through the use of microfluidics, where cells are cultured on top of the PCTE membrane and electroporated with macromolecules actively flowing beneath. Using properties of laminar flow and a high Péclet number, parallel streams of two different cargo types with minimal mixing between them allow for spatially patterned delivery.

We aimed to investigate the impact of active flow on HT-1080 cells on a nanoporous membrane by first determining the optimal flow rate to minimize mixing between parallel streams, then assessing the effect of fluid shear stress on cell viability, morphology, and adherence during and after flow. Parallel fluid streams were withdrawn with a syringe pump at varying velocities until 1.67 mm/s, or 20 µL/min was deemed optimal with a mixing area of 20% or less. This flow rate was run for five minutes with cell media beneath adhered HT-1080 cells, with images taken every minute during flow with Hoechst and Calcein AM stains to assess cell adherence and morphology under fluid shear stress of 1.00 dyn/cm2. Viability imaging with Ethidium Homodimer and Hoechst was performed 24 hours later.

Eccentricity measures the extent to which a cell’s shape deviates from a perfect circle, which has a value of 0. When under stress, cells can start to ball up (decreasing eccentricity), so we used eccentricity as a measurement for assessing one aspect of cell health during active flow. HT-1080 cells have an epithelial morphology and a slightly elongated shape. The mean eccentricity values for both the flow and no flow conditions remained within 0.68-0.72 and cell count remained constant, so cells were not immediately damaged by flow. After 24 hours, viability for the control and flow conditions was between 66-75% per device, with no significant difference between the conditions.

Parallel flow at a velocity of 1.67 mm/s was established underneath HT-1080 cells adhered to a PCTE membrane, and the resulting shear stress of 1.00 dyn/cm2 caused minimal disruption to cell viability when compared to the control. This means that cell death was likely caused by factors other than flow, though future experiments will include more imaging steps within the 24 hours after an experiment to view cell health over a longer time period. Since viability in all conditions was below 75%, we will next make improvements to our device design and protocol to increase viability to over 90%. Having established parallel flow, this system can be electroporated to pattern a flow of two macromolecular streams into cells.