Design and Fabrication of Micro/Nano Sensors and Actuators.

A micro-electromechanical system (MEMS) is a micro device or system that utilizes large-scale integrated circuit manufacturing technology and microfabrication technology to integrate microsensors, micro-actuators, microstructures, signal processing and control circuits, power supplies, and communication interfaces into one or more chips [...].

Desmosomal Cadherin Tension Loss in Pemphigus Vulgaris Mediated by the Inhibition of Active RhoA at Cell-Cell Adhesions.

Binding of autoantibodies to keratinocyte surface antigens, primarily desmoglein 3 (Dsg3) of the desmosomal complex, leads to the dissociation of cell-cell adhesion in the blistering disorder pemphigus vulgaris (PV). After the initial disassembly of desmosomes, cell-cell adhesions actively remodel in association with the cytoskeleton and focal adhesions. Growing evidence highlights the role of adhesion mechanics and mechanotransduction at cell-cell adhesions in this remodeling process, as their active participation may direct autoimmune pathogenicity. However, a large part of the biophysical transformations after antibody binding remains underexplored. Specifically, it is unclear how tension in desmosomes and cell-cell adhesions changes in response to antibodies, and how the altered tensional states translate to cellular responses. Here, we showed a tension loss at Dsg3 using fluorescence resonance energy transfer (FRET)-based tension sensors, a tension loss at the entire cell-cell adhesion, and a potentially compensatory increase in junctional traction force at cell-extracellular matrix adhesions after PV antibody binding. Further, our data indicate that this tension loss is mediated by the inhibition of RhoA at cell-cell contacts, and the extent of RhoA inhibition may be crucial in determining the severity of pathogenicity among different PV antibodies. More importantly, this tension loss can be partially restored by altering actomyosin based cell contractility. Collectively, these findings provide previously unattainable details in our understanding of the mechanisms that govern cell-cell interactions under physiological and autoimmune conditions, which may open the window to entirely new therapeutics aimed at restoring physiological balance to tension dynamics that regulates the maintenance of cell-cell adhesion.

Active viscoelastic models for cell and tissue mechanics.

Living cells are out of equilibrium active materials. Cell-generated forces are transmitted across the cytoskeleton network and to the extracellular environment. These active force interactions shape cellular mechanical behaviour, trigger mechano-sensing, regulate cell adaptation to the microenvironment and can affect disease outcomes. In recent years, the mechanobiology community has witnessed the emergence of many experimental and theoretical approaches to study cells as mechanically active materials. In this review, we highlight recent advancements in incorporating active characteristics of cellular behaviour at different length scales into classic viscoelastic models by either adding an active tension-generating element or adjusting the resting length of an elastic element in the model. Summarizing the two groups of approaches, we will review the formulation and application of these models to understand cellular adaptation mechanisms in response to various types of mechanical stimuli, such as the effect of extracellular matrix properties and external loadings or deformations.

Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery.

Porous substrate electroporation (PSEP) is a promising new method for intracellular delivery, yet fundamentals of PSEP are not well understood, especially the intermediate processes leading to delivery. PSEP is an electrical method, yet the relationship between PSEP and electrical impedance remains underexplored. In this study, a device capable of measuring impedance and performing PSEP is developed and the changes in transepithelial electrical impedance (TEEI) are monitored. These measurements show TEEI increases following PSEP, unlike other electroporation methods. The authors then demonstrate how cell culture conditions and electrical waveforms influence this response. More importantly, TEEI response features are correlated with viability and delivery efficiency, allowing prediction of outcomes without fluorescent cargo, imaging, or image processing. This label-free delivery also allows improved temporal resolution of transient processes following PSEP, which the authors expect will aid PSEP optimization for new cell types and cargos.

Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery.

Porous substrate electroporation (PSEP) is a promising new method for intracellular delivery, yet fundamentals of the PSEP delivery process are not well understood, partly because most PSEP studies rely solely on imaging for evaluating delivery. Although effective, imaging alone limits understanding of intermediate processes leading to delivery. PSEP is an electrical process, so electrical impedance measurements naturally complement imaging for PSEP characterization. In this study, we developed a device capable of measuring impedance and performing PSEP and we monitored changes in transepithelial electrical impedance (TEEI). Our measurements show TEEI increases following PSEP, unlike other electroporation methods. We then demonstrated how cell culture conditions and electrical waveforms influence this response. More importantly, we correlated TEEI response features with viability and delivery efficiency, allowing prediction of outcomes without fluorescent cargo, imaging, or image processing. This label-free delivery also allows improved temporal resolution of transient processes following PSEP, which we expect will aid PSEP optimization for new cell types and cargos.

Two-Photon Polymerized Shape Memory Microfibers: A New Mechanical Characterization Method in Liquid.

Two-photon polymerization (TPP) has been widely used to create 3D micro- and nanoscale scaffolds for biological and mechanobiological studies, which often require the mechanical characterization of the TPP fabricated structures. To satisfy physiological requirements, most of the mechanical characterizations need to be conducted in liquid. However, previous characterizations of TPP fabricated structures were all conducted in air due to the limitation of conventional micro- and nanoscale mechanical testing methods. In this study, we report a new experimental method for testing the mechanical properties of TPP-printed microfibers in liquid. The experiments show that the mechanical behaviors of the microfibers tested in liquid are significantly different from those tested in air. By controlling the TPP writing parameters, the mechanical properties of the microfibers can be tailored over a wide range to meet a variety of mechanobiology applications. In addition, it is found that, in water, the plasticly deformed microfibers can return to their pre-deformed shape after tensile strain is released. The shape recovery time is dependent on the size of microfibers. The experimental method represents a significant advancement in mechanical testing of TPP fabricated structures and may help release the full potential of TPP fabricated 3D tissue scaffold for mechanobiological studies.

Characterizing nuclear morphology and expression of eNOS in vascular endothelial cells subjected to a continuous range of wall shear stress magnitudes and directionality.

Complex patterns of hemodynamic wall shear stress occur in regions of arterial branching and curvature. Areas within these regions can be highly susceptible to atherosclerosis. Although many studies have characterized the response of vascular endothelial cells to shear stress in a categorical manner, our study herein addresses the need of characterizing endothelial behaviors over a continuous range of shear stress conditions that reflect the extensive variations seen in the vasculature. We evaluated the response of human umbilical vein endothelial cell monolayers to orbital flow at 120, 250, and 350 revolutions per minute (RPM) for 24 and 72 h. The orbital shaker model uniquely provides a continuous range of shear stress conditions from low and multidirectional at the center of each well of a culture plate to high and unidirectional at the periphery. We found distinct patterns of endothelial nuclear area, nuclear major and minor diameters, nuclear aspect ratio, and expression of endothelial nitric oxide synthase over this range of shear conditions and relationships were fit with linear and, where appropriate, power functions. Nuclear area was particularly sensitive with increases in the low and multidirectional WSS region that incrementally decreased as WSS became higher in magnitude and more unidirectional over the radius of the cell layers. The patterns of all endothelial behaviors exhibited high correlations (positive and negative) with metrics of shear stress magnitude and directionality that have been shown to strongly associate with atherosclerosis. Our findings demonstrate the exquisite sensitivity of these endothelial behaviors to incremental changes in shear stress magnitude and directionality, and provide critical quantitation of these relationships for predicting the susceptibility of an arterial segment to diseases such as atherosclerosis, particularly within complex flow environments in the vasculature such as around bifurcations.

YAP mechanotransduction under cyclic mechanical stretch loading for mesenchymal stem cell osteogenesis is regulated by ROCK.

While yes-associated protein (YAP) is now recognized as a potent mechanosensitive transcriptional regulator to affect cell growth and differentiation including the osteogenic transcription of mesenchymal stem cells (MSCs), most studies have reported the YAP mechanosensing of static mechanophysical cues such as substrate stiffness. We tested MSC response to dynamic loading, i.e., cyclic mechanical stretching, and assessed YAP mechanosensing and resultant MSC osteogenesis. We showed that cyclic stretching at 10% strain and 1 Hz frequency triggered YAP nuclear import in MSCs. YAP phosphorylation at S127 and S397, which is required for YAP cytoplasmic retention, was suppressed by cyclic stretch. We also observed that anti-YAP-regulatory Hippo pathway, LATS phosphorylation, was significantly decreased by stretch. We confirmed the stretch induction of MSC osteogenic transcription and differentiation, and this was impaired under YAP siRNA suggesting a key role of YAP dynamic mechanosensing in MSC osteogenesis. As an underlying mechanism, we showed that the YAP nuclear transport by cyclic stretch was abrogated by ROCK inhibitor, Y27632. ROCK inhibitor also impaired the stretch induction of F-actin formation and MSC osteogenesis, thus implicating the role of the ROCK-F-actin cascade in stretch-YAP dynamic mechanosensing-MSC osteogenesis. Our results provide insight into bone tissue engineering and skeletal regenerative capacity of MSCs especially as regards the role of dynamic mechanical loading control of YAP-mediated MSC osteogenic transcription.

A multi-material platform for imaging of single cell-cell junctions under tensile load fabricated with two-photon polymerization.

We previously reported a single-cell adhesion micro tensile tester (SCAµTT) fabricated from IP-S photoresin with two-photon polymerization (TPP) for investigating the mechanics of a single cell-cell junction under defined tensile loading. A major limitation of the platform is the autofluorescence of IP-S, the photoresin for TPP fabrication, which significantly increases background signal and makes fluorescent imaging of stretched cells difficult. In this study, we report the design and fabrication of a new SCAµTT platform that mitigates autofluorescence and demonstrate its capability in imaging a single cell pair as its mutual junction is stretched. By employing a two-material design using IP-S and IP-Visio, a photoresin with reduced autofluorescence, we show a significant reduction in autofluorescence of the platform. Further, by integrating apertures onto the substrate with a gold coating, the influence of autofluorescence on imaging is almost completely mitigated. With this new platform, we demonstrate the ability to image a pair of epithelial cells as they are stretched up to 250% strain, allowing us to observe junction rupture and F-actin retraction while simultaneously recording the accumulation of over 800 kPa of stress in the junction. The platform and methodology presented here can potentially enable detailed investigation of the mechanics of and mechanotransduction in cell-cell junctions and improve the design of other TPP platforms in mechanobiology applications.

Rho/ROCK mechanosensor in adipocyte stiffness and traction force generation.

It is increasingly recognized that interaction of adipose cells with extracellular mechanophysical milieus may play a role in regulating adipogenesis and differentiated adipocyte function and such interaction can be mediated by the mechanics of adipose cells. We measured the stiffness and traction force of adipose cells and examined the role of Rho/ROCK, the upstream effector of actin cytoskeletal contractility, in affecting these mechanical properties. Cellular Young's modulus obtained from atomic force microscopy (AFM) was significantly reduced by ROCK inhibitor (Y-27632) but elevated by Rho activator (CN01), for both preadipocytes and differentiated adipocytes. Immunofluorescent imaging suggested this could be attributed to the changes in Rho/ROCK-induced stressed actin filament formation. AFM also confirmed that differentiated adipocytes had higher stiffness than preadipocytes. On the other hand, traction force microscopy (TFM) revealed differentiated adipocytes exerted lower traction forces than preadipocytes. Traction forces of both preadipocytes and adipocytes were decreased by ROCK inhibition, but not significantly altered by Rho activation. Notably, an increasing trend of traction force with respect to cell spreading area was detected, and this trend was substantially amplified by Rho activation. Such traction force-cell area correlation was an order-of-magnitude smaller for differentiated adipocytes relative to preadipocytes, potentially due to disrupted force transmission through cytoskeleton-focal adhesion linkage by lipid droplets. Our work provides new data evidencing the Rho/ROCK control in adipose cell mechanics, laying the groundwork for adipocyte mechanotransduction studies on adipogenesis and adipose tissue remodeling.

High-Throughput DNA Tensioner Platform for Interrogating Mechanical Heterogeneity of Single Living Cells.

Cell mechanical forces play fundamental roles in regulating cellular responses to environmental stimulations. The shortcomings of conventional methods, including force resolution and cellular throughput, make them less accessible to mechanical heterogeneity at the single-cell level. Here, a DNA tensioner platform is introduced with high throughput (>10 000 cells per chip) and pN-level resolution. A microfluidic-based cell array is trapped on "hairpin-structured" DNA tensioners that enable transformation of the mechanical information of living cells into fluorescence signals. By using the platform, one can identify enhanced mechanical forces of drug-resistant cells as compared to their drug-sensitive counterparts, and mechanical differences between metastatic tumor cells in pleural effusion and nonmetastatic histiocytes. Further genetic analysis traces two genes, VEGFA and MINK1, that may play deterministic roles in regulating mechanical heterogeneities. In view of the ubiquity of cells' mechanical forces in the extracellular microenvironment (ECM), this platform shows wide potential to establish links of cellular mechanical heterogeneity to genetic heterogeneity.

An equivalent circuit model for localized electroporation on porous substrates.

In vitro intracellular delivery is a fundamental challenge with no widely adopted methods capable of both delivering to millions of cells and controlling that delivery to a high degree of accuracy. One promising method is porous substrate electroporation (PSEP), where cells are cultured on porous substrates and electric fields are used to permeabilize discrete portions of the cell membrane for delivery. A major obstacle to the widespread use of PSEP is a poor understanding of the various impedances that constitute the system, including the impedances of the porous substrate and the cell monolayer, and how these impedances are influenced by experimental parameters. In response, we used impedance measurements to develop an equivalent circuit model that closely mimics the behavior of each of the main components of the PSEP system. This circuit model reveals for the first time the distribution of voltage across the electrode-electrolyte interface impedances, the channels of the porous substrate, the cell monolayer, and the transmembrane potential during PSEP. We applied sample waveforms through our model to understand how waveforms can be improved for future studies. Our model was validated from intracellular delivery of protein using PSEP.

Spatially Guided Construction of Multilayered Epidermal Models Recapturing Structural Hierarchy and Cell-Cell Junctions.

A current challenge in three-dimensional (3D) bioprinting of skin equivalents is to recreate the distinct basal and suprabasal layers and to promote their direct interactions. Such a structural arrangement is essential to establish 3D stratified epidermis disease models, such as for the autoimmune skin disease pemphigus vulgaris (PV), which targets the cell-cell junctions at the interface of the basal and suprabasal layers. Inspired by epithelial regeneration in wound healing, we develop a method that combines 3D bioprinting and spatially guided self-reorganization of keratinocytes to recapture the fine structural hierarchy that lies in the deep layers of the epidermis. Here, keratinocyte-laden fibrin hydrogels are bioprinted to create geographical cues, guiding dynamic self-reorganization of cells through collective migration, keratinocyte differentiation and vertical expansion. This process results in a region of self-organized multilayers (SOMs) that contain the basal to suprabasal transition, marked by the expressed levels of different types of keratins that indicate differentiation. Finally, we demonstrate the reconstructed skin tissue as an in vitro platform to study the pathogenic effects of PV and observe a significant difference in cell-cell junction dissociation from PV antibodies in different epidermis layers, indicating their applications in the preclinical test of possible therapies.

Modulation of Mechanical Stress Mitigates Anti-Dsg3 Antibody-Induced Dissociation of Cell-Cell Adhesion.

It is becoming increasingly clear that mechanical stress in adhesive junctions plays a significant role in dictating the fate of cell-cell attachment under physiological conditions. Targeted disruption of cell-cell junctions leads to multiple pathological conditions, among them the life-threatening autoimmune blistering disease pemphigus vulgaris (PV). The dissociation of cell-cell junctions by autoantibodies is the hallmark of PV, however, the detailed mechanisms that result in tissue destruction remain unclear. Thus far, research and therapy in PV have focused primarily on immune mechanisms upstream of autoantibody binding, while the biophysical aspects of the cell-cell dissociation process leading to acantholysis are less well studied. In work aimed at illuminating the cellular consequences of autoantibody attachment, it is reported that externally applied mechanical stress mitigates antibody-induced monolayer fragmentation and inhibits p38 MAPK phosphorylation activated by anti-Dsg3 antibody. Further, it is demonstrated 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. The study elevates understanding of the mechanism of acantholysis in PV and shifts the paradigm of PV disease development from a focus solely on immune pathways to highlight the key role of physical transformations at the target cell.

Characterization of the strain-rate-dependent mechanical response of single cell-cell junctions.

Cell-cell adhesions are often subjected to mechanical strains of different rates and magnitudes in normal tissue function. However, the rate-dependent mechanical behavior of individual cell-cell adhesions has not been fully characterized due to the lack of proper experimental techniques and therefore remains elusive. This is particularly true under large strain conditions, which may potentially lead to cell-cell adhesion dissociation and ultimately tissue fracture. In this study, we designed and fabricated a single-cell adhesion micro tensile tester (SCAµTT) using two-photon polymerization and performed displacement-controlled tensile tests of individual pairs of adherent epithelial cells with a mature cell-cell adhesion. Straining the cytoskeleton-cell adhesion complex system reveals a passive shear-thinning viscoelastic behavior and a rate-dependent active stress-relaxation mechanism mediated by cytoskeleton growth. Under low strain rates, stress relaxation mediated by the cytoskeleton can effectively relax junctional stress buildup and prevent adhesion bond rupture. Cadherin bond dissociation also exhibits rate-dependent strengthening, in which increased strain rate results in elevated stress levels at which cadherin bonds fail. This bond dissociation becomes a synchronized catastrophic event that leads to junction fracture at high strain rates. Even at high strain rates, a single cell-cell junction displays a remarkable tensile strength to sustain a strain as much as 200% before complete junction rupture. Collectively, the platform and the biophysical understandings in this study are expected to build a foundation for the mechanistic investigation of the adaptive viscoelasticity of the cell-cell junction.

Nanosensors for single cell mechanical interrogation.

The occurrence and development of many diseases are accompanied and sometimes dictated by the destruction of biomechanical homeostasis. For instance, cancer cells and normal cells show different cellular mechanical forces phenotypes, as the proliferation and invasion ability of cancer cells is often related to the changes in mechanical force in the tumor. With single cell analysis, variations in mechanics within a cell population can be detected and analyzed, opening new dimensions in the study of cancer. Nanosensor design for interrogation of cell mechanics is an interdisciplinary area bridging over cell biology, mechanics, and micro/nanotechnology. In this tutorial review, we give insight into the background and technical innovation of currently available methods for mechanical analysis of cells. First, we discuss the mechanism of mechanical changes in the development and progression of cancer that shows the feasibility of mechanical sensors in cancer cell detection. Next, we summarize the principle, progress, and essential problems of common technologies for cell force measurement, including single molecule force spectroscopy and elastic substrate-sensors. Following that, we discuss novel micro and nano-scale mechanical sensors and their applications in single cell level biological analysis. At last, we elaborate on the remaining issues and trends of the cellular mechanical sensors.

High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery.

In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow-through microfluidics, engineered substrates, and automated probe-based systems from the perspective of throughput and control. Special attention is paid to a particularly promising method of electroporation using micro/nanochannel based porous substrates, which expose small patches of cell membrane to permeabilizing electric field. Porous substrate electroporation parameters discussed include system design, cells and cargos used, transfection efficiency and cell viability, and the electric field and its effects on molecular transport. The review concludes with discussion of potential new innovations which can arise from specific aspects of porous substrate-based electroporation platforms and high throughput, high control methods in general.

Facile Production of Large-Area Cell Arrays Using Surface-Assembled Microdroplets.

Techniques that enable the spatial arrangement of living cells into defined patterns are broadly applicable to tissue engineering, drug screening, and cell-cell investigations. Achieving large-scale patterning with single-cell resolution while minimizing cell stress/damage is, however, technically challenging using existing methods. Here, a facile and highly scalable technique for the rational design of reconfigurable arrays of cells is reported. Specifically, microdroplets of cell suspensions are assembled using stretchable surface-chemical patterns which, following incubation, yield ordered arrays of cells. The microdroplets are generated using a microfluidic-based aerosol spray nozzle that enables control of the volume/size of the droplets delivered to the surface. Assembly of the cell-loaded microdroplets is achieved via mechanically induced coalescence using substrates with engineered surface-wettability patterns based on extracellular matrices. Robust cell proliferation inside the patterned areas is demonstrated using standard culture techniques. By combining the scalability of aerosol-based delivery and microdroplet surface assembly with user-defined chemical patterns of controlled functionality, the technique reported here provides an innovative methodology for the scalable generation of large-area cell arrays with flexible geometries and tunable resolution.

An Active Biomechanical Model of Cell Adhesion Actuated by Intracellular Tensioning-Taxis.

Cell adhesion to the extracellular matrix (ECM) is highly active and plays a crucial role in various physiological functions. The active response of cells to physicochemical cues has been universally discovered in multiple microenvironments. However, the mechanisms to rule these active behaviors of cells are still poorly understood. Here, we establish an active model to probe the biomechanical mechanisms governing cell adhesion. The framework of cells is modeled as a tensional integrity that is maintained by cytoskeletons and extracellular matrices. Active movement of the cell model is self-driven by its intrinsic tendency to intracellular tensioning, defined as tensioning-taxis in this study. Tensioning-taxis is quantified as driving potential to actuate cell adhesion, and the traction forces are solved by our proposed numerical method of local free energy adaptation. The modeling results account for the active adhesion of cells with dynamic protruding of leading edge and power-law development of mechanical properties. Furthermore, the morphogenesis of cells evolves actively depending on actin filaments alignments by a predicted mechanism of scaling and directing traction forces. The proposed model provides a quantitative way to investigate the active mechanisms of cell adhesion and holds the potential to guide studies of more complex adhesion and motion of cells coupled with multiple external cues.

The Role of Fluid Shear and Metastatic Potential in Breast Cancer Cell Migration.

During the migration of cancer cells for metastasis, cancer cells can be exposed to fluid shear conditions. We examined two breast cancer cell lines, MDA-MB-468 (less metastatic) and MDA-MB-231 (more metastatic), and a benign MCF-10A epithelial cell line for their responsiveness in migration to fluid shear. We tested fluid shear at 15 dyne/cm2 that can be encountered during breast cancer cells traveling through blood vessels or metastasizing to mechanically active tissues such as bone. MCF-10A exhibited the least migration with a trend of migrating in the flow direction. Intriguingly, fluid shear played a potent role as a trigger for MDA-MB-231 cell migration, inducing directional migration along the flow with significantly increased displacement length and migration speed and decreased arrest coefficient relative to unflowed MDA-MB-231. In contrast, MDA-MB-468 cells were markedly less migratory than MDA-MB-231 cells, and responded very poorly to fluid shear. As a result, MDA-MB-468 cells did not exhibit noticeable difference in migration between static and flow conditions, as was distinct in root-mean-square (RMS) displacement-an ensemble average of all participating cells. These may suggest that the difference between more metastatic MDA-MB-231 and less metastatic MDA-MB-468 breast cancer cells could be at least partly involved with their differential responsiveness to fluid shear stimulatory cues. Our study provides new data in regard to potential crosstalk between fluid shear and metastatic potential in mediating breast cancer cell migration.