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Research Thrust 1: Single Cell-Cell Mechanical Interrogation and Strain Rate-Dependent Mechanical Behavior Characterization. To study cell-cell junction mechanics and mechanotransduction at the single-cell level, we developed a polymeric microstructure to strain the mutual junction of a single cell pair while simultaneously recording the junction stress. We call this platform a single-cell adhesion micro tensile tester (SCAµTT). To the best of our knowledge, SCAµTT is the first platform that allows in situ interrogation of the stress-strain characteristics of a mature cell-cell junction through defined strain and strain rate, promoting a paradigm shift in the mechanical characterization of cell-cell adhesions. With this platform, we pioneered the study of the strain-rate dependent mechanical behavior of cell-cell junctions.

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Research Thrust 2: Effect of Mechanical Load on Cell-Cell Adhesion Pathology. Combined with a cell monolayer mechanical stimulation platform and a single cell pair interrogation platform, we demonstrated a protective mechanism of mechanical load against antibody (Ab)-induced cell-cell adhesion loss in an autoimmune skin disease model, pemphigus vulgaris (PV). We show that mechanical stress applied externally to cell monolayers enhances cell contractility via RhoA activation and promotes the strengthening of cortical actin, which ultimately mitigates antibody-induced cell-cell dissociation. Our study reinforces the key regulatory role of sequential mechanical stress variations in the maintenance and disruption of cell-cell adhesion that continues to shift the paradigm of PV disease development from a focus solely on immune pathways to a deeper understanding of the key role in cyto-mechanics in pathology at the target cell level.

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Research Thrust 3: Single Cell Array Assembly Using Liquid Microdroplets. Single cell analysis needs to be performed in a high throughput system. We engineered an innovative system to enable single-cell resolution patterning at high throughput. We developed a facile and highly scalable technique for the rational design of reconfigurable arrays of cells, which we call micro-assembly of cells-in-droplets (µACD). In µACD, microdroplets of cell suspensions were assembled using stretchable surface-chemical patterns which, following incubation, yielded ordered arrays of cells. By combining the scalability of aerosol-based delivery and microdroplet surface assembly with user defined chemical patterns of controlled functionality, our technique provides an innovative methodology for the scalable generation of large-area cell arrays with flexible geometries and tunable resolution down to the single cell level.

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Research Thrust 4: Mechanism of Porous Substrate Electroporation for Electrokinetic Transport and Single Cell Intracellular Delivery. Porous substrate electroporation (PSEP) allows localized permeabilization of small patches of cell membrane for intracellular delivery. The electrokinetic transport of molecules during the PSEP process has never been fully understood, mainly because the electrical potentials across the cell membrane and the micro-/nanochannels of the porous substrate are unknown. We have developed an equivalent circuit model that closely mimics the behavior of each of the main components of the PSEP system from measured impedance data, and this model was validated through electroporation experiments. Our circuit model reveals, for the first time, the distribution of voltage across the faradaic impedances of the electrodes, the cell monolayer on the porous substrates and through the micro-/nanochannels of the porous substrate during PSEP using experimental data. Our study allows us to evaluate the voltage-dependent electrokinetics involved in transport through the porous substrate and how these electrokinetics change with channel size.

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Research Facilities. We host a range of state-of-the-art instruments and tools including: micro/nano manipulators for cell manipulation, atomic force microscopy for high resolution imaging and mechanical characterization, optical microscopy for fluorescence imaging, and a host of electronics including micro-controllers, patch-clamp amplifiers, oscilloscopes and more. The nanoscience core at UNL hosts a number of state-of-the-art instruments: scanning electron microscopy, transmission electron microscopy, atomic force microscopy, and nanofabrication clean rooms.

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