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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.

The reproduction and survival of fish are often negatively affected by the construction of dams and other hydroelectric projects, which cut off their migratory routes. Building effective fish passage facilities that allow fish to pass through dams smoothly alleviates the negative impact of hydroelectric projects on the ecological environment, thus protecting the diversity of aquatic species and preventing the extinction of indigenous fish. Vertical slit fishways are highly effective, but turbulence inside the fishway pools directly affects fish passage. In this study, the large-eddy simulation framework is used to capture the vortex characteristics in the interior of vertical slit fishway pools, and the volume of fluid method is applied to simulate the free surface. The independence of the grid is assessed by the large-eddy simulation quality index, and the simulation results are compared with experimental acoustic Doppler velocimetry data. This work characterizes the vortex flow field inside the vertical slit fishway using the Q-criterion, Omega method, and Liutex vortex identification method. The results show that the vortex structure inside the fishway pool has obvious three-dimensional characteristics and vortex structure varies within the different fishway pool chambers. The analysis and comparison of the three different vortex identification methods show that the vortex structure captured by the Liutex method is more consistent with the actual motion pattern of the fishway water flow.

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.

The ability to generate chemical and mechanical gradients on chips is important for either creating biomimetic designs or enabling high-throughput assays. However, there is still a significant knowledge gap in the generation of mechanical and chemical gradients in a single device. In this study, we developed gradient-generating microfluidic circuits with integrated microchambers to allow cell culture and to introduce chemical and mechanical gradients to cultured cells. A chemical gradient is generated across the microchambers, exposing cells to a uniform concentration of drugs. The embedded microchamber also produces a mechanical gradient in the form of varied shear stresses induced upon cells among different chambers as well as within the same chamber. Cells seeded within the chambers remain viable and show a normal morphology throughout the culture time. To validate the effect of different drug concentrations and shear stresses, doxorubicin is flowed into chambers seeded with skin cancer cells at different flow rates (from 0 to 0.2 μL/min). The experimental results show that increasing doxorubicin concentration (from 0 to 30 μg/mL) within chambers not only prohibits cell growth but also induces cell death. In addition, the increased shear stress (0.005 Pa) at high flow rates poses a synergistic effect on cell viability by inducing cell damage and detachment. Moreover, the ability of the device to seed cells in a 3D microenvironment was also examined and confirmed. Collectively, the study demonstrates the potential of microchamber-embedded microfluidic gradient generators in 3D cell culture and high-throughput drug screening.

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.

Atomic force microscopy (AFM) based nanotechnology has been widely implemented in various fields for decades in light of its overwhelming advantages, such as nanometer spatial resolution, adaptability to liquid ambient, and various nanomechanical/electrical metrological approaches. It is noted that though AFM possesses imaging capability up to nanometer resolution, it is hard to achieve nanometer level positioning precision due to the existing system variability, especially the thermal drift, which distorts AFM images through relatively long capturing time. Since an AFM image is typically utilized as a global reference map to navigate its tip to the desired locations for precise measurement and manipulation, the system variability distorted image will definitely diversify the experimental results. Therefore, it is necessary to characterize the positioning variability for better experimental results evaluation and decision-making. Although various approaches were proposed to evaluate AFM positioning error, to our best knowledge, there is little research about characterizing its positioning variability precisely and systematically. In this study, we present a universal metrological approach to quantitatively measure AFM tip locating variability by developing a featureless spiral local scan strategy together with the non-vector space (NVS) navigation approach. As a demonstration, the proposed nanometrology was conducted on a specific AFM platform to unravel its positioning property.

We developed an Adaptive Reference-Digital Image Correlation (AR-DIC) method that enables unbiased and accurate mechanics measurements of moving biological tissue samples. We applied the AR-DIC analysis to a spontaneously beating cardiomyocyte (CM) tissue, and could provide correct quantifications of tissue displacement and strain for the beating CMs utilizing physiologically-relevant, sarcomere displacement length-based contraction criteria. The data were further synthesized into novel spatiotemporal parameters of CM contraction to account for the CM beating homogeneity, synchronicity, and propagation as holistic measures of functional myocardial tissue development. Our AR-DIC analyses may thus provide advanced non-invasive characterization tools for assessing the development of spontaneously contracting CMs, suggesting an applicability in myocardial regenerative medicine.

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.

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.

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. Ministry of Education (MOE) Nanyang Technological University Submitted/Accepted version We acknowledge the funding support from the NSF(Awards 1826135 and 1936065), the NIH National Institutes of GeneralMedical Sciences P20GM113126 (Nebraska Center for Integrated Biomolec-ular Communication), P20GM104320 (Nebraska Center for the Prevention ofObesity Diseases), P30GM127200 (Nebraska Center for Nanomedicine),1U54GM115458-01 (Great Plains IDeA-CTR), and R15AR072959. We acknowl-edge funding support from the Nebraska Collaborative Initiative andEPSCoR FIRST award. Design and fabrication of the TPP structures wereconducted at the Center for Nanophase Materials Sciences at Oak RidgeNational Laboratory, which is a Department of Energy Office of Science UserFacility. C.H. would also like to acknowledge financial support from NanyangTechnological University (Startup Grant M4082352.050) and the Ministry ofEducation, Singapore, under its AcademicResearch Fund Tier1 (M4012229.050).M.D. acknowledges partial support from NIH R01HL154150.

Microfluidic devices have shown capabilities for precise cell manipulation and analysis for decades. In recent years, the improvement of microfabrication technology and the introduction of new materials have boosted innovation on single-cell electroporation on microfluidic devices. This has opened the door for gene transfection and drug delivery with high efficiency and cellular safety in the exploration of cell biology and life science. In this chapter, we introduce the basic mechanism and state of the art of electroporation techniques based on microfluidic devices. We first discuss three types of electroporation systems for high-throughput cell transfection, including bulk electroporation, tubular microfluidic electroporation, and a localized electroporation device. Next, we shed light on cell manipulation techniques integrated on microfluidic electroporation devices to achieve higher efficiency and safer electroporation. Several methods for cell manipulation and their applications in electroporation for biomedicine are introduced.

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.

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.