Investigation of Single-Cell and Blood-Brain Barrier Mechanics after Electroporation and in Primary Brain Cancers

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Virginia Tech


Cell-level and tissue-level mechanical properties are key to healthy biological functions, and many diseases and disorder arise or progress due to altered cell and tissue mechanics. Pulse electric field (PEFs), which employ intense external electric fields to cause electroporation, a phenomenon characterized by increased cell membrane permeability, also can cause significant changes to cell and tissue mechanics. Here, we investigate the mechanics of brain and brain cancer cells, specifically focusing on how PEFs impact cell mechanics and PEF-induced blood-brain barrier disruption. In our first study, we investigate single-cell mechanical disruption of glioblastoma cells after reversible electroporation using Nanonet Force Microscopy (NFM). A precise network of extracellular-matrix mimicking nanofibers enabled cell attachment and contraction, resulting in measurable fiber deflections. Cell contractile forces were shown to be temporarily disrupted after reversible electroporation, in an orientation and field-dependent manner. Furthermore, we found that cell response is often a multi-stage process involving a cell-rounding stage, biphasic stage, and a cell re-spreading stage. Additionally, cell viability post-PEFs was orientation-dependent. In another study, we investigated the mechanical properties of brain cancer for various-grade glioma cells (healthy astrocytes, grade II, grade III, and grade IV (glioblastoma) cells). A microfluidic constriction channel caused cell deformation as cells, driven by hydrostatic pressure, entered a narrow constriction. Finite element models of cell deformation and a neural network were used to convert experimental results (cell entry time and cell elongation within the channel) into elastic modulus values (kPa). We found that the that low-grade glioma cells showed higher stiffnesses compared to healthy and grade IV glioma cells, which both showed similar values. These results warrant future studies to investigate these trends further. PEFs can induce Blood-brain barrier (BBB) disruption, an effect we studied using a multiplexed, PDMS microdevice. A monolayer of human cerebral endothelial cells on a semi-permeable membrane was used to model the BBB, and permeability was assessed by the diffusion of a fluorescent dye from an upper to lower channel. A custom tapered channel and branching channel design created a linear gradient in the electric field within the device that enabled six electric field strengths to be tested at once against two unexposed (control) channels. Normalization of permeability by the control channels significantly removed experimental noise. We found that after high-frequency bipolar irreversible electroporation (HFIRE) electric pulses, permeability transiently increased within the first hour after electroporation, in a voltage- and pulse-number dependent manner. However, we found significant electrofusion events after pulsing at high voltages, which reduced monolayer permeability below baseline values. This device enables efficient exploration of a wide range of electroporation parameters to identify the optimal conditions for blood-brain barrier disruption. In another blood-brain barrier study, we incorporate dense, polystyrene nanofiber networks to create ultra-thin, ultra-porous basement-membrane-mimics for In vitro blood-brain barrier models. Fiber networks are fabricated using the non-electrospinning Spinneret-based Tunable Engineered Parameters (STEP) technique. Endothelial cells cultured on one side of the fiber network are in close contact with supporting cell types (pericytes) cultured on the backside of the fibers. Contact-orientation co-cultures have been shown to increase blood-brain barrier integrity, and our nanofiber networks increase the physiological realism of basement-membrane mimics for improve modeling. Finally, we investigate how cell viability post-electroporation is impacted by cell morphology. The impact of cell morphology (shape and cytoskeletal structure) on cell survival after electroporation is not well understood. Linking specific morphological characteristics with cell susceptibility to electroporation will enhance fundamental knowledge and will be widely useful for improving electroporation techniques where cell viability is desirable (gene transfection, electrofusion, electrochemotherapy) or where cell viability is undesirable (tumor ablation, cardiac ablation). Precise control of cell shape and orientation enabled by nanofiber scaffolds provides a convenient and expedient platform for investigating a wide variety of factors (morphological and experimental) on cell viability. Altogether, these investigations shed new light on cell mechanical changes due to disease and pulsed electric fields, and suggest opportunities for improving brain cancer therapies.



Pulsed electric fields, blood-brain barrier, electroporation, microfluidics, mechanobiology, cytoskeleton