Biomedical Microsystems

High-throughput, rapid and non-invasive readouts of tissue health in microfluidic kidney co-culture models would expand their capabilities for pre-clinical assessment of drug-induced nephrotoxicity. Here, we demonstrate a technique for monitoring steady state oxygen levels in PREDICT96-O₂, a high-throughput organ-on-chip platform with integrated optical-based oxygen sensors, for evaluation of drug-induced nephrotoxicity in a human microfluidic co-culture model of the kidney proximal tubule (PT). Oxygen consumption measurements in PREDICT96-O₂ detected dose and time-dependent injury responses of human PT cells to cisplatin, a drug with known toxic effects in the PT. The injury concentration threshold of cisplatin decreased exponentially from 19.8 µM after 1 day to 2.3 µM following a clinically relevant exposure duration of 5 days. Additionally, oxygen consumption measurements resulted in a more robust and expected dose-dependent injury response over multiple days of cisplatin exposure compared to colorimetric-based cytotoxicity readouts. The results of this study demonstrate the utility of steady state oxygen measurements as a rapid, non-invasive, and kinetic readout of drug-induced injury in high-throughput microfluidic kidney co-culture models.

Measurement of cell metabolism in moderate-throughput to high-throughput organ-on-chip (OOC) systems would expand the range of data collected for studying drug effects or disease in physiologically relevant tissue models. However, current measurement approaches rely on fluorescent imaging or colorimetric assays that are focused on endpoints, require labels or added substrates, and lack real-time data. Here, we integrated optical-based oxygen sensors in a high-throughput OOC platform and developed an approach for monitoring cell metabolic activity in an array of membrane bilayer devices. Each membrane bilayer device supported a culture of human renal proximal tubule epithelial cells on a porous membrane suspended between two microchannels and exposed to controlled, unidirectional perfusion and physiologically relevant shear stress for several days. For the first time, we measured changes in oxygen in a membrane bilayer format and used a finite element analysis model to estimate cell oxygen consumption rates (OCRs), allowing comparison with OCRs from other cell culture systems. Finally, we demonstrated label-free detection of metabolic shifts in human renal proximal tubule cells following exposure to FCCP, a drug known for increasing cell oxygen consumption, as well as oligomycin and antimycin A, drugs known for decreasing cell oxygen consumption. The capability to measure cell OCRs and detect metabolic shifts in an array of membrane bilayer devices contained within an industry standard microtiter plate format will be valuable for analyzing flow-responsive and physiologically complex tissues during drug development and disease research.

Background: Extremely preterm (EP) birth is associated with higher risks of perinatal white matter (WM) injury, potentially causing abnormal neurologic and neurocognitive outcomes. MRI biomarkers distinguishing individuals with and without neurologic disorder guide research on EP birth antecedents, clinical correlates, and prognoses. Purpose: To compare multiparametric quantitative MRI (qMRI) parameters of EP-born adolescents with autism spectrum disorder, cerebral palsy, epilepsy, or cognitive impairment (ie, atypically developing) with those without (ie, neurotypically developing), characterizing sex-stratified brain development. Materials and Methods: This prospective multicenter study included individuals aged 14–16 years born EP (Extremely Low Gestational Age Newborns–Environmental Influences on Child Health Outcomes Study, or ELGAN-ECHO). Participants underwent 3.0-T MRI evaluation from 2017 to 2019. qMRI outcomes were compared for atypically versus neurotypically developing adolescents and for girls versus boys. Sex-stratified multiple regression models were used to examine associations between spatial entropy density (SE_d) and T1, T2, and cerebrospinal fluid (CSF)–normalized proton density (nPD), and between CSF volume and T2. Interaction terms modeled differences in slopes between atypically versus neurotypically developing adolescents. Results: A total of 368 adolescents were classified as 116 atypically (66 boys) and 252 neurotypically developing (125 boys) participants. Atypically versus neurotypically developing girls had lower nPD (mean, 557 10 × percent unit [pu] ± 46 [SD] vs 573 10 × pu ± 43; P = .04), while atypically versus neurotypically developing boys had longer T1 (814 msec ± 57 vs 789 msec ± 82; P = .01). Atypically developing girls versus boys had lower nPD and shorter T2 (eg, in WM, 557 10 × pu ± 46 vs 580 10 × pu ± 39 for nPD [P = .006] and 86 msec ± 3 vs 88 msec ± 4 for T2 [P = .003]). Atypically versus neurotypically developing boys had a more moderate negative association between T1 and SEd (slope, –32.0 msec per kB/cm³ [95% CI: –49.8, –14.2] vs –62.3 msec per kB/cm³ [95% CI: –79.7, –45.0]; P = .03). Conclusion: Atypically developing participants showed sexual dimorphisms in the cerebrospinal fluid–normalized proton density (nPD) and T2 of both white matter (WM) and gray matter. Atypically versus neurotypically developing girls had lower WM nPD, while atypically versus neurotypically developing boys had longer WM T1 and more moderate T1 associations with microstructural organization in WM.

Central venous catheters (CVCs) are implanted in the majority of dialysis patients despite increased patient risk due to thrombotic occlusion and biofilm formation. Current solutions remain ineffective at preventing these complications and treatment options are limited and often harmful. We present further analysis of the previously proposed water infused surface protection (WISP) technology, an active method to reduce protein adsorption and effectively disrupt adsorbed protein sheaths on the inner surface of CVCs. A WISP CVC is modeled by a hollow fiber membrane (HFM) in a benchtop device which continuously infuses a saline solution across the membrane wall into the blood flow, creating a blood-free boundary layer at the lumen surface. Total protein adsorption is measured under various experimental conditions to further test WISP performance. The WISP device shows reduced protein adsorption as blood and WISP flow rates increase (P < 0.040) with up to a 96% reduction in adsorption over the no WISP condition. When heparin is added to the WISP flow, protein adsorption (0.097[+0.035/-0.055] µg/mm²) is reduced when compared to both bolus administration and nondoped WISP, 0.406(+0.056/-0.065) µg/mm² (P = 0.001) and 0.191 (+0.076/-0.126) (P = 0.029), respectively. Additionally, when heparinized WISP is applied to a preadsorbed protein layer, 0.375(+0.114/-0.164) µg/mm², it displays the ability to reduce the previously-adsorbed protein, 0.186((+0.058/-0.084) µg/mm² (P = 0.0012), suggesting aptitude for intermittent treatments. The WISP technology not only shows the ability to reduce protein adsorption, but also the ability to remove preadsorbed material by effectively delivering drugs to the point of adsorption; functionalities that could greatly improve clinical outcomes.

Protein adhesion in central venous catheters (CVCs) leads to fibrin sheath formation, the precursor to thrombotic and biofilm-related CVC failures. Advances in material properties and surface coatings do not completely prevent fibrin sheath formation and post-formation treatment options are limited and expensive. We propose water infused surface protection (WISP), an active method for prevention of fibrin sheath formation on CVCs, which creates a blood-free boundary layer on the inner surface of the CVC, limiting blood contact with the CVC lumen wall. A hollow fiber membrane (HFM) in a benchtop device served as a CVC testing model to demonstrate the WISP concept. Porcine blood was pumped through the HFM while phosphate buffered saline (PBS) was infused through the HFM wall, creating the WISP boundary layer. Protein adherences on model CVC surfaces were measured and imaged. Analytical and finite volume lubrication models were used to justify the assumption of a blood-free boundary layer. We found a 92.2% reduction in average adherent protein density when WISP is used, compared with our model CVC without WISP flow. Lubrication models matched our experimental pressure drop measurements suggesting that a blood-free boundary layer was created. The WISP technique also provides a novel strategy for drug administration for biofilm treatment. Reduction in adherent protein indicates a restriction on long-term fibrin sheath and biofilm formation making WISP a promising technology which improves a wide range of vascular access treatments.

This paper reports novel MEMS and NEMS-based fabrication processes for biocompatible, hollow cylindrical ferromagnetic structures for potential use as contrast agents for magnetic resonance imaging (MRI). Compared to previous works on Ni-based cylindrical-nanoshells and Fe-based double-disk particles, biocompatibility and yield issues were strongly considered in this development of a simplified fabrication process incorporating iron oxide thin films. The novel, simplified fabrication processes developed herein yield robust, reproducible fabrication methodologies for the further development of this new class of MRI contrast agents. Specifically, both micron- and nano-scale hollow cylindrical agents were successfully fabricated, the size regimes of which enable a wide array of potential imaging applications. The use of top-down engineering approaches to MRI contrast agent design such as reported herein offers the capacity for multiplexed imaging which may dramatically potentiate the capabilities of MRI imaging.

This paper reports the fabrication and characterization of composite hydrogel particles composed of poly(ethylene glycol) diacrylate (PEG-DA)-based hydrogels and x-ray attenuating payloads. The top–down fabrication method employed herein is demonstrated to yield composite hydrogel particles of varying size and shape for use as computed tomography (CT) imaging contrast agents. Characterization of the materials properties of the PEG-DA hydrogels was undertaken, demonstrating tunable mechanical properties of composite hydrogels based on hydrogel composition and UV cross-linking time. Analyses of the leakage rates of a conventional iodine-based small molecular contrast agent as well as a nanoparticulate x-ray attenuating material from the PEG-DA hydrogels were undertaken. In contradistinction to clinically available iodinated CT contrast agents, as well as recently developed nanoparticulate CT contrast agents, the approach presented herein yields an engineering flexibility to the design of CT contrast agents which may be leveraged to optimize this class of agents to a wide array of specific imaging and sensing applications.

Recently, a novel class of magnetic resonance imaging (MRI) contrast agents developed using top-down microfabrication approaches has been reported. To realize the full capacity of this potentially paradigm-shifting approach to MRI contrast agent design, the integration of biocompatible materials with tunable magnetic properties was sought. To this end, deposition techniques yielding iron oxide thin films with a large range of readily tunable saturation magnetic polarization were developed using reactive sputtering under various conditions. Following the characterization of their chemical compositions and crystalline structures, the iron oxide thin films were subsequently utilized in the fabrication of size and shape specific magnetic double-disk microparticles, yielding the advantages of this new class of MRI contrast agents, including multiplexing capability, diffusion-driven signal amplification, and functional imaging capacity. The integration of iron oxides into this class of fabricated contrast agents offers several distinct advantages, including biocompatibility and the additional degree of freedom in the design of these agents achieved by the tunability of the iron oxide thin film magnetism, both of which are critical features in further optimizing these agents.

Models of reabsorptive barriers require both a means to provide realistic physiologic cues to and quantify transport across a layer of cells forming the barrier. Here we have topographically-patterned porous membranes with several user-defined pattern types. To demonstrate the utility of the patterned membranes, we selected one type of pattern and applied it to a membrane to serve as a cell culture support in a microfluidic model of a renal reabsorptive barrier. The topographic cues in the model resemble physiological cues found in vivo while the porous structure allows quantification of transport across the cell layer. Sub-micron surface topography generated via hot-embossing onto a track-etched polycarbonate membrane, fully replicated topographical features and preserved porous architecture. Pore size and shape were analyzed with SEM and image analysis to determine the effect of hot embossing on pore morphology. The membrane was assembled into a bilayer microfluidic device and a human kidney proximal tubule epithelial cell line (HK-2) and primary renal proximal tubule epithelial cells (RPTEC) were cultured to confluency on the membrane. Immunofluorescent staining of both cell types revealed protein expression indicative of the formation of a reabsorptive barrier responsive to mechanical stimulation: ZO-1 (tight junction), paxillin (focal adhesions) and acetylated α-tubulin (primary cilia). HK-2 and RPTEC aligned in the direction of ridge/groove topography of the membrane in the device, evidence that the device has mechanical control over cell response. This topographically-patterned porous membrane provides an in vitro platform on which to model reabsorptive barriers with meaningful applications for understanding biological transport phenomenon, underlying disease mechanisms, and drug toxicity.

Physiologically-representative and well-controlled in vitro models of human tissue provide a means to safely, accurately, and rapidly develop therapies for disease. Current in vitro models do not possess appropriate levels of cell function, resulting in an inaccurate representation of in vivo physiology. Mechanical parameters, such as sub-micron topography and flow-induced shear stress (FSS), influence cell functions such as alignment, migration, differentiation and phenotypic expression. Combining, and independently controlling, biomaterial surface topography and FSS in a cell culture device would provide a means to control cell function resulting in more physiologically-representative in vitro models of human tissue. Here we develop the Microscale Tissue Modeling Device (MTMD) which couples a topographically-patterned substrate with a microfluidic chamber to control both topographic and FSS cues to cells. Cells from the human renal proximal tubule cell line HK-2 were cultured in the MTMD and exposed to topographic patterns and several levels of FSS simultaneously. Results show that the biomaterial property of surface topography and FSS work in concert to elicit cell alignment and influence tight junction (TJ) formation, with topography enhancing cell response to FSS. By administering independently-controlled mechanical parameters to cell populations, the MTMD creates a more realistic in vitro model of human renal tissue.

In this work, a time-domain fluorescence spectroscopy technique is developed to characterize thermophysical properties of polymers. The method is based on fluorescence thermometry of materials under periodic pulse heating. In the characterization, a continuous laser (405 nm) is modulated with adjustable periodic heating and fluorescence excitation. The temperature rise at sample surface due to laser heating is probed from simultaneous fluorescence spectrum. Thermal diffusivity can be determined from the relationship between normalized temperature rise and the duration of laser heating. To verify this technique, thermal diffusivity of a polymer material (PVC) is characterized as 1.031 × 10^-7 m²/s, agreeing well with reference data. Meanwhile, thermal conductivity can be obtained by the hot plate method. Then, both steady and unsteady thermophysical properties are available. Quenching effect of fluorescence signal in our measurement can be ignored, as validated by longtime laser heating experiments. The uncertainty induced by uniformity of laser heating is negligible as analyzed through numerical simulations. This non-destructive fluorescence-based technique does not require exact value about laser absorption and calibration experiment for temperature coefficient of fluorescence signals. Considering that most polymers can excite sound fluorescence signal, this method can be well applied to thermal characterization of polymer-based film or bulk materials.

The mechanical properties of the local microenvironment may have important influence on the fate and function of adult tissue progenitor cells, altering the regenerative process. This is particularly critical following a myocardial infarction, in which the normal, compliant myocardial tissue is replaced with fibrotic, stiff scar tissue. In this study, we examined the effects of matrix stiffness on adult cardiac side population (CSP) progenitor cell behavior. Ovine and murine CSP cells were isolated and cultured on polydimethylsiloxane substrates, replicating the elastic moduli of normal and fibrotic myocardium. Proliferation capacity and cell cycling were increased in CSP cells cultured on the stiff substrate with an associated reduction in cardiomyogeneic differentiation and accelerated cell ageing. In addition, culture on stiff substrate stimulated upregulation of extracellular matrix and adhesion proteins gene expression in CSP cells. Collectively, we demonstrate that microenvironment properties, including matrix stiffness, play a critical role in regulating progenitor cell functions of endogenous resident CSP cells. Understanding the effects of the tissue microenvironment on resident cardiac progenitor cells is a critical step toward achieving functional cardiac regeneration.

A generic experimental procedure is presented in this work to enhance the electrical responses of polydimethylsiloxane (PDMS) through incorporation of conducting polymer nanowires, while maintaining the desirable mechanical flexibility of PDMS. The conducting polypyrrole (PPy) nanowires are synthesized using a template method. The dielectric constants of the composites are characterized by impedance measurements, and the effect of nanowire concentration is investigated by the percolation theory. Using a continuous hyperbolic tangent function, critical volume fraction is estimated to be 9.6 vol%, at which an 85-fold enhancement in the dielectric constants is achieved. The viscoelastic properties of the composites are characterized by the stress relaxation nanoindentation tests, and the effect of nanowire concentration on the elastic modulus of composites is found to deviate significantly from the Wang–Pyrz model at the critical volume fraction. The tunable multifunctionality of PDMS composites that possess significantly enhanced electrical and moderate viscoelastic responses is desirable for many sensing and actuation applications.

Polydimethylsiloxane (PDMS) micropillar-based biotransducers are extensively used in cellular force measurements. The accuracy of these devices relies on the appropriate material characterization of PDMS and modeling to convert the micropillar deformations into the corresponding forces. Cellular contraction is often accompanied by oscillatory motion, the frequency of which ranges in several hertz. In this paper, we developed a methodology to calculate the cellular contraction forces in the frequency domain with improved accuracy. The contraction data were first expressed as a Fourier series. Subsequently, we measured the complex modulus of PDMS using a dynamic nanoindentation technique. An improved method for the measurement of complex modulus was developed with the use of a flat punch indenter. The instrument dynamics was characterized, and the full contact region was identified. By incorporating both the Fourier series of contraction data and the complex modulus function, the cellular contraction force was calculated by finite-element analysis (FEA). The difference between the Euler beam formula and the viscoelastic FEA was discussed. The methodology presented in this work is anticipated to benefit the material characterization of other soft polymers and complex biological behavior in the frequency domain.

Polymeric deformable sensor arrays have been employed to measure cellular forces and offered insights into the study of cellular mechanics. Previous studies have been focused on using transducers in static domain and assumed elastic beam theory as the force conversion model. Neglecting the inherent viscoelastic behavior of polydimethylsiloxane and low aspect ratios of the sensor arrays compromised the accuracy of these devices. In this work, a more in-depth viscoelastic Timoshenko beam model was developed incorporating dynamic cellular forces. We studied chemically stimulated contractions of cardiac myocytes and found that the loading rate has a considerable influence on the sensitivity of the sensor arrays.

Electroactive conducting polymers (CPs) have been frequently used for fabricating bending actuators. To model this type of actuation, the traditional double-layer beam bending theory was implemented by neglecting the thickness of the thin intermediate metal layers for the sake of simplification. However, this common assumption has not been carefully validated and the associated errors have not been well acknowledged. In this work, a generic multilayer bending model was introduced to account for the actuators consisting of an arbitrary number of layers. Our model found the bending curvature, strain, stress, and in particular work density of the multilayer actuator as explicit functions of the thickness and modulus of each individual layer. The thickness of metals and conducting polymers were controlled in thermal evaporation and electrochemical synthesis, respectively. The modulus of polypyrrole (PPy), the conducting polymer used in this work, was determined within our model by the bending curvature measured using the charge-coupled device (CCD). This gave a modulus of our electrochemically synthesized PPy of 80 MPa, corresponding to an actuation strain of 2% in our model. It was concluded that neglecting the intermediate metal layers would lead to substantial errors. For instance, using a PPy/Au/Kapton trilayer actuator, a 5% error or below in strain can only be found if the Au layer is one thousand times thinner than Kapton. To enhance the actuation, a PPy/Pt/PVDF/Pt/PPy five-layer actuator has been often used. In this case, even if the Pt layer was reduced to 10 nm, our predicted error of neglecting the two metal layers would be 12.59%. Our results showed that the work density, chosen to measure the overall performance of the actuator, was highly sensitive to the modulus of the substrate polymer layer so that it was generally desirable of using a soft polymer substrate. With the multilayer bending model, we intend to provide an accurate and reliable tool for systematically analyzing the bending behavior and performance of the CP-based actuators.

Polydimethylsiloxane (PDMS)-based micropillars (or microcantilevers) have been used as bio-transducers for measuring cellular forces on the order of pN to µN. The measurement accuracy of these sensitive devices depends on appropriate modeling to convert the micropillar deformations into the corresponding reaction forces. The traditional approach to calculating the reaction force is based on the Euler beam theory with consideration of a linear elastic slender beam for the micropillar. However, the low aspect ratio in geometry of PDMS micropillars does not satisfy the slender beam requirement. Consequently, the Timoshenko beam theory, appropriate for a beam with a low aspect ratio, should be used. In addition, the inherently time-dependent behavior in PDMS has to be considered for accurate force conversion. In this paper, the Timoshenko beam theory, along with the consideration of viscoelastic behavior of PDMS, was used to model the mechanical response of micropillars. The viscoelastic behavior of PDMS was characterized by stress relaxation nanoindentation using a circular flat punch. A correction procedure was developed to determine the load–displacement relationship with consideration of ramp loading. The relaxation function was extracted and described by a generalized Maxwell model. The bending of rectangular micropillars was performed by a wedge indenter tip. The viscoelastic Timoshenko beam formula was used to calculate the mechanical response of the micropillar, and the results were compared with measurement data. The calculated reaction forces agreed well with the experimental data at three different loading rates. A parametric study was conducted to evaluate the accuracy of the viscoelastic Timoshenko beam model by comparing the reaction forces calculated from the elastic Euler beam, elastic Timoshenko beam and viscoelastic Euler beam models at various aspect ratios and loading rates. The extension of modeling from the elastic Euler beam theory to the viscoelastic Timoshenko beam theory has improved the accuracy for the conversion of the PDMS micropillar deformations to forces, which will benefit the polymer-based micro bio-transducer applications.

Polydimethylsiloxane (PDMS) is an important polymeric material widely used in bio-MEMS devices such as micropillar arrays for cellular mechanical force measurements. The accuracy of such a measurement relies on choosing an appropriate material constitutive model for converting the measured structural deformations into corresponding reaction forces. However, although PDMS is a well-known viscoelastic material, many researchers in the past have treated it as a linear elastic material, which could result in errors of cellular traction force interpretation. In this paper, the mechanical properties of PDMS were characterized by using uniaxial compression, dynamic mechanical analysis, and nanoindentation tests, as well as finite element analysis (FEA). A generalized Maxwell model with the use of two exponential terms was used to emulate the mechanical behavior of PDMS at room temperature. After we found the viscoelastic constitutive law of PDMS, we used it to develop a more accurate model for converting deflection data to cellular traction forces. Moreover, in situ cellular traction force evolutions of cardiac myocytes were demonstrated by using this new conversion model. The results presented by this paper are believed to be useful for biologists who are interpreting similar physiological processes.

Polydimethylsiloxane (PDMS) microcantilevers have been used as force sensors for studying cellular mechanics by converting their displacements to cellular mechanical forces. However, PDMS is an inherently viscoelastic material and its elastic modulus changes with loading rates and elapsed time. Therefore, the traditional approach to calculating cellular mechanical forces based on elastic mechanics can result in errors. This letter reports a more in-depth method for viscoelastic characterization, modeling, and analysis associated with the bending behavior of the PDMS microcantilevers. A viscoelastic force conversion model was developed and validated by proof-of-principle bending tests.

The mapping of traction forces is crucial to understanding the means by which cells regulate their behavior and physiological function to adapt to and communicate with their local microenvironment. To this end, polymeric micropillar arrays have been used for measuring cell traction force. However, the small scale of the micropillar deflections induced by cell traction forces results in highly inefficient force analyses using conventional optical approaches; in many cases, cell forces may be below the limits of detection achieved using conventional microscopy. To address these limitations, the moiré phenomenon has been leveraged as a visualization tool for cell force mapping due to its inherent magnification effect and capacity for whole-field force measurements. This Letter reports an optomechanical cell force sensor, namely, a double-sided micropillar array (DMPA) made of poly(dimethylsiloxane), on which one side is employed to support cultured living cells while the opposing side serves as a reference pattern for generating moiré patterns. The distance between the two sides, which is a crucial parameter influencing moiré pattern contrast, is predetermined during fabrication using theoretical calculations based on the Talbot effect that aim to optimize contrast. Herein, double-sided micropillar arrays were validated by mapping mouse embryo fibroblast contraction forces and the resulting force maps compared to conventional microscopy image analyses as the reference standard. The DMPA-based approach precludes the requirement for aligning two independent periodic substrates, improves moiré contrast, and enables efficient moiré pattern generation. Furthermore, the double-sided structure readily allows for the integration of moiré-based cell force mapping into microfabricated cell culture environments or lab-on-a-chip devices.

Microsystems are providing key advances in studying single-cell mechanical behaviours. The mechanical interaction of cells with their extracellular matrix is fundamentally important for cell migration, division, phagocytes and apoptosis. As the displacement and scales of cellular phenomena is comparable to optical wavelength, optical metrology offers superior resolution and real-time imaging capabilities to measure cell forces and subcellular behaviour as compared to its traditional counterparts. This review Letter discusses the principles, formation and methodological aspects of building opto-mechanical systems in studying cell forces. The authors report current advances of various opto-mechanical systems in studying different aspects of cell mechanics.

Microsystems are providing key advances in studying single cell mechanical behavior. The mechanical interaction of cells with their extracellular matrix is fundamentally important for cell migration, division, phagocytosis and aptoptosis. This review reports the development of microsystems on studying cell forces. Microsystems provide advantages of studying single cells since the scale of cells is on the micron level. The components of microsystems provide culture, loading, guiding, trapping and on chip analysis of cellular mechanical forces. This paper gives overviews on how MEMS are advancing in the field of cell biomechno sensory systems. It presents different materials, and mode of studying cell mechanics. Finally, we comment on the future directions and challenges on the state of art techniques.

Microfabricated polymer transducers have been developed to study cell mechanics. The key principle is to quantify the deformations on the sensor arrays induced by cell contractions and convert them into force distributions. The simplifications in deformation measurements come from the basic assumption that the deformation is solely attributed to cell contractions triggered by chemical or electrical stimuli. The diffraction moiré fringes via two polymer gratings provide whole field evolutions of distortion/strain on soft-lithography fabricated substrates. We found that the moiré patterns are able to decouple predistortions which were traditionally thought to be solely caused by cell contractile forces.

The mapping of contraction forces developed from cells to their extracellular matrix is crucial to understanding how cells regulate their physiological function to adapt to their living environment and cellular processes. This paper reports a novel cell contractility mapping transducer utilizing moiré patterns as a visual and quantitative tool. Coherent light diffracted from two closely placed microfabricated periodic substrates is capable of mapping cell contraction forces via mapping the in-plane displacement on the sample substrate. By integrating cell culture environment and automated Fourier-based fringe analysis, the moiré pattern generated through microfabricated periodic substrates enables the mapping of cell contraction force distribution through phase changes encoded in carrier moiré fringe patterns. We demonstrated utilizing the transducer to map cardiac myocyte contraction under electric stimulation and vascular smooth muscle cell contractility evolutions triggered by agonist. Given the unique properties of optical moiré techniques (i.e., their automatic displacement and strain contouring and their magnification effect for small displacements), this new approach would be an improvement over existing techniques since it can be integrated with the existing engineered substrates and provide a direct contour of cell forces and fast detection of abnormal cell contractions.

Abnormal vascular cell contractile performance is a hallmark of cardiovascular diseases. Conventional cell force measurement technique requires individually tracking the sensing units and complex computation efforts for further studying cell contractility. We developed instead a robust and simple compact optical moiré system that measures phase changes encoded in carrier moiré patterns generated from two layers of gratings. Cell mechanics study including cell contractile forces and stress and strain distributions during normal and abnormal cell contractions can thus be conveniently analyzed. The distinct signals from moiré patterns in longitudinal and transverse directions revealed abnormal cell mechanical contractility linked to cardiovascular disease.

Abnormal vascular smooth muscle cell (VSMC) contraction plays an important role in vascular diseases. The RhoA/ROCK signaling pathway is now well recognized to mediate vascular smooth muscle contraction in response to vasoconstrictors by inhibiting myosin phosphatase (MLCP) activity and increasing myosin light chain phosphorylation. Two ROCK isoforms, ROCK1 and ROCK2, are expressed in many tissues, yet the isoform-specific roles of ROCK1 and ROCK2 in vascular smooth muscle and the mechanism of ROCK-mediated regulation of MLCP are not well understood. In this study, ROCK2, but not ROCK1, bound directly to the myosin binding subunit of MLCP, yet both ROCK isoforms regulated MLCP and myosin light chain phosphorylation. Despite that both ROCK1 and ROCK2 regulated MLCP, the ROCK isoforms had distinct and opposing effects on VSMC morphology and ROCK2, but not ROCK1, had a predominant role in VSMC contractility. These data support that although the ROCK isoforms both regulate MLCP and myosin light chain phosphorylation through different mechanisms, they have distinct roles in VSMC function.

Cells alter their shape and morphology and interact with their surrounding environment. Mechanical forces developed by cells to their surrounding environments are fundamental to many physiological processes, such as cell growth, division, migration and apoptosis. In this paper, a novel optical Moiré based biomechanol force sensor was developed for cell traction force mapping. We utilized coherent laser beams to illuminate periodic polymeric substrates where isolated cells were cultured. We demonstrated one-dimensional and two-dimensional traction force mapping via optical Moiré for both cardiac myocytes and vascular smooth muscle cells. The magnification effect of the Moiré fringe pattern permits a real time monitoring of the mechanical interaction between isolated cells and their underlying periodic polymeric structures.

This letter reports an approach for cell contraction force mapping by utilizing optical moiré effect. We cultured living cells on patterned polymer substrates and studied the diffraction moiré patterns. We found that the flexible moiré patterns generated on the periodic substrates are capable of mapping cell contraction force evolution in whole field. We demonstrated one- and two-dimensional force mappings in vascular cells. Due to moiré magnification, this imaging approach can provide a versatile visual tool for mapping the cell-substrate interactions in living cells.

In this article, a PDMS microfluidic immunosensor integrated with specific antibody immobilized alumina nanoporous membrane was developed for rapid detection of foodborne pathogens Escherichia coli O157:H7 and Staphylococcus aureus with electrochemical impedance spectrum. Firstly, antibodies to the targeted bacteria were covalently immobilized on the nanoporous alumina membranes via self assembled (3-glycidoxypropyl)trimethoxysilane (GPMS) silane. Then, the impedance spectrum was recorded for bacteria detection ranging from 1 Hz to 100 kHz. The maximum impedance amplitude change for these two food pathogens was around 100 Hz. This microfluidic immunosensor based on nanoporous membrane impedance spectrum could achieve rapid bacteria detection within 2 h with a high sensitivity of 10² CFU/ml. Cross-bacteria experiments for E. coli O157:H7 and S. aureus were also explored to testify the specificity. The results showed that impedance amplitude at 100 Hz had a significant reduction in binding of bacteria when the membrane was exposed to non-specific bacteria.

Cardiomyocyte death caused by proinflammatory cytokines, such as Tumor necrosis factor α (TNF-α), is one of the hot topics in cardiovascular research. TNF-α can induce multiple cell processes that are dependent on the treatment time although the long-term treatment definitely leads to cell death. The ability to intervene in cell death will be invaluable to reveal the effects of short-term TNF-α treatment to cardiomyocytes. However, a real-time monitoring technique is needed to guide the intervention of cell responses. In this work, we employed the impedance-sensing technique to real-time monitor the equivalent cell–substrate distance of cardiomyocytes via electrochemical impedance spectroscopy (EIS) and electrical cell–substrate impedance sensing (ECIS). In the stabilized cardiomyocyte culture, the sustained TNF-α treatment caused strengthened cell adhesion in the first 2 h which was followed by the transition to cell detachment afterwards. Considering cell detachment was an early morphological evidence of cell death, we removed TNF-α from the cardiomyocyte culture before the transition to achieve the intervention of cell responses. The result of this intervention showed that cell adhesion was continuously strengthened before and after the removal of TNF-α, indicating the short-term treated cardiomyocytes did not undergo death processes. It was also demonstrated in TUNEL and TBE tests that the percentages of apoptosis and cell death were both lowered.

Deregulated cardiomyocyte death is a critical risk factor in a variety of cardiovascular diseases. Although various assays have been developed to detect cell responses during cell death, the capability of monitoring cell detachment will enhance the understanding of death processes by providing instant information at its early phase. In this work, we developed an impedance-sensing assay for real-time monitoring of cardiomyocyte death induced by tumor necrosis factor-α based on recording the change in cardiomyocyte adhesion to extracellular matrix. Electrochemical impedance spectroscopy was employed in impedance data processing, followed by calibration with the electrical cell-substrate impedance-sensing technique. The adhesion profile of cardiomyocytes undergoing cell death processes was recorded as the time course of equivalent cell-substrate distance. The cell detachment was detected with our assay and proved related to cell death in the following experiments, indicating its advantage against the conventional assays, such as Trypan blue exclusion. An optimal concentration of tumor necrosis factor-α (20 ng/mL) was determined to induce cardiomyocyte apoptosis rather than the combinative cell death of necrosis and apoptosis by comparing the concentration-related adhesion profiles. The cardiomyocytes undergoing apoptosis experienced an increase of cell-substrate distance from 59.1 to 89.2 nm within 24 h. The early change of cell adhesion was proved related to cardiomyocyte apoptosis in the following TUNEL test at t = 24 h, which suggested the possibility of early and noninvasive detection of cardiomyocyte apoptosis.

The cell−substrate distance is a direct indicator of cell adhesion to extracellular matrix which is indispensable in cell culture. A real-time monitoring approach can provide a detailed profile of cell adhesion, so that enables the detecting of adhesion-related cell behavior. In this work, we report a novel real-time impedance-based method to record the adhesion profile of cardiomyocyte, overcoming its inscrutability due to the primary culture. Microfabricated biosensors are applied in cardiomyocyte culture after characterizing the cell-free system. Cyclic frequency scanning data of cell-related impedance are generated and automatically fit into the equivalent circuit model, which is established using electrochemical impedance spectroscopy. The data are displayed as the alteration of normalized cell−substrate distance and the essential parameters for manual electric cell−substrate impedance sensing calibration of absolute distance. The time course displays a significant decline in the equivalent cell−substrate distance, from 155.8 to 60.2 nm in the first 20 h of cardiomyocyte culture. Furthermore, the cardiomyocytes cultured in long-term medium and short-term medium (ACCT) for 10 h exhibit distinct difference in adhesion rate as well as cell−substrate distance (72 vs 68 nm).

Cardiac hypertrophy is the heart’s response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress that may be regulated by growth factor such as Endothelin-1 (ET-1). The majority of existing techniques to monitor hypertrophy in vitro are based on florescence probes designed to show morphological and biochemical alterations indicative of cardiomyocyte hypertrophy. In this work, a new cardiomyocyte-based impedance sensing system with the assistance of dielectrophoresis (DEP) cell concentration is developed to monitor the dynamics process of ET-1 induced cardiomyocyte hypertrophy. This device can increase the sensitivity of the impedance system and also has the potential to reduce the time for detection by a significant factor.

Cardiac tissue engineering has evolved as a potential therapeutic approach to assist cardiac regeneration. Controlling the preferential cell orientation of engineered heart tissues is a key issue in cardiac tissue engineering. Here, we present a novel method to construct a model-engineered cardiac tissue-like structure with anisotropic properties. Our analysis shows that the electro-torque which acts on a cylindrical or rod shape cell is zero whenever the electric field is aligned with one of its principal axes. With the interdigitated–castellated microelectrodes, the induction of dielectrophoresis and electro-orientation can accumulate cells and form a tissue-like structure with orientation along the ac electric field. Both experiments and analysis indicate that a large orientation torque and force can be achieved with appropriate frequency and low conductive medium. Finally, we report basic structural and biophysical anisotropy of electro-oriented structure through electromechanical experiments.

Cardiac hypertrophy is an established and independent risk factor for the development of heart failure and sudden cardiac death. At the level of individual cardiac myocytes (heart muscle cells), the cell morphology alters (increase in cell size and myofibrillar re-organization) and protein synthesis is activated. In this paper, a novel cardiomyocyte-based impedance sensing system with the assistance of dielectrophoresis cell concentration is reported to monitor the dynamic process of endothelin-1-induced cardiomyocyte hypertrophy. A dielectrophoresis (DEP) microfluidic device is fabricated capable of concentrating cells from a dilute sample to form a confluent cell monolayer on the surface of microelectrodes. This device can increase the sensitivity of the impedance system and also has the potential to reduce the time for detection by a significant factor. To examine the feasibility of this impedance sensing system, cardiomyocytes are treated with endothelin-1 (ET-1), a known hypertrophic agent. ET-1 induces a continuous rise in cardiomyocyte impedance, which we interpret as strengthening of cellular attachments to the surface substrate. An equivalent circuit model is introduced to fit the impedance spectrum to fully understand the impedance sensing system.

Recently, the ability to create engineered heart tissues with a preferential cell orientation has gained much interest. Here, we present a novel method to construct a cardiac myocyte tissue-like structure using a combination of dielectrophoresis and electro-orientation via a microfluidic chip. The device includes a top home-made silicone chamber containing microfluidic channels and bottom integrated microelectrodes which are patterned on a glass slide to generate dielectrophoresis force and orientation torque. Using the interdigitated-castellated microelectrodes, the induction of a mutually attractive dielectrophoretic force between cardiac myocytes can lead to cells moving close to each other and forming a tissue-like structure with orientation along the alternating current (ac) electric field between the microelectrode gaps. Both experiments and analysis indicate that a large orientation torque and force can be achieved by choosing an optimal frequency around 2 MHz and decreasing the conductivity of medium to a relatively low level. Finally, electromechanical experiments and biopolar impedance measurements were performed to demonstrate the structural and functional anisotropy of electro-oriented structure.

In this article, a PDMS microfluidic immunosensor integrated with specific antibody immobilized alumina nanoporous membrane was developed for rapid detection of foodborne pathogens Escherichia coli O157:H7 and Staphylococcus aureus with electrochemical impedance spectrum. Firstly, antibodies to the targeted bacteria were covalently immobilized on the nanoporous alumina membranes via self assembled (3-glycidoxypropyl)trimethoxysilane (GPMS) silane. Then, the impedance spectrum was recorded for bacteria detection ranging from 1 Hz to 100 kHz. The maximum impedance amplitude change for these two food pathogens was around 100 Hz. This microfluidic immunosensor based on nanoporous membrane impedance spectrum could achieve rapid bacteria detection within 2 h with a high sensitivity of 10² CFU/ml. Cross-bacteria experiments for E. coli O157:H7 and S. aureus were also explored to testify the specificity. The results showed that impedance amplitude at 100 Hz had a significant reduction in binding of bacteria when the membrane was exposed to non-specific bacteria.

Cardiomyocyte death caused by proinflammatory cytokines, such as Tumor necrosis factor α (TNF-α), is one of the hot topics in cardiovascular research. TNF-α can induce multiple cell processes that are dependent on the treatment time although the long-term treatment definitely leads to cell death. The ability to intervene in cell death will be invaluable to reveal the effects of short-term TNF-α treatment to cardiomyocytes. However, a real-time monitoring technique is needed to guide the intervention of cell responses. In this work, we employed the impedance-sensing technique to real-time monitor the equivalent cell–substrate distance of cardiomyocytes via electrochemical impedance spectroscopy (EIS) and electrical cell–substrate impedance sensing (ECIS). In the stabilized cardiomyocyte culture, the sustained TNF-α treatment caused strengthened cell adhesion in the first 2 h which was followed by the transition to cell detachment afterwards. Considering cell detachment was an early morphological evidence of cell death, we removed TNF-α from the cardiomyocyte culture before the transition to achieve the intervention of cell responses. The result of this intervention showed that cell adhesion was continuously strengthened before and after the removal of TNF-α, indicating the short-term treated cardiomyocytes did not undergo death processes. It was also demonstrated in TUNEL and TBE tests that the percentages of apoptosis and cell death were both lowered.

Deregulated cardiomyocyte death is a critical risk factor in a variety of cardiovascular diseases. Although various assays have been developed to detect cell responses during cell death, the capability of monitoring cell detachment will enhance the understanding of death processes by providing instant information at its early phase. In this work, we developed an impedance-sensing assay for real-time monitoring of cardiomyocyte death induced by tumor necrosis factor-α based on recording the change in cardiomyocyte adhesion to extracellular matrix. Electrochemical impedance spectroscopy was employed in impedance data processing, followed by calibration with the electrical cell-substrate impedance-sensing technique. The adhesion profile of cardiomyocytes undergoing cell death processes was recorded as the time course of equivalent cell-substrate distance. The cell detachment was detected with our assay and proved related to cell death in the following experiments, indicating its advantage against the conventional assays, such as Trypan blue exclusion. An optimal concentration of tumor necrosis factor-α (20 ng/mL) was determined to induce cardiomyocyte apoptosis rather than the combinative cell death of necrosis and apoptosis by comparing the concentration-related adhesion profiles. The cardiomyocytes undergoing apoptosis experienced an increase of cell-substrate distance from 59.1 to 89.2 nm within 24 h. The early change of cell adhesion was proved related to cardiomyocyte apoptosis in the following TUNEL test at t = 24 h, which suggested the possibility of early and noninvasive detection of cardiomyocyte apoptosis.

The cell−substrate distance is a direct indicator of cell adhesion to extracellular matrix which is indispensable in cell culture. A real-time monitoring approach can provide a detailed profile of cell adhesion, so that enables the detecting of adhesion-related cell behavior. In this work, we report a novel real-time impedance-based method to record the adhesion profile of cardiomyocyte, overcoming its inscrutability due to the primary culture. Microfabricated biosensors are applied in cardiomyocyte culture after characterizing the cell-free system. Cyclic frequency scanning data of cell-related impedance are generated and automatically fit into the equivalent circuit model, which is established using electrochemical impedance spectroscopy. The data are displayed as the alteration of normalized cell−substrate distance and the essential parameters for manual electric cell−substrate impedance sensing calibration of absolute distance. The time course displays a significant decline in the equivalent cell−substrate distance, from 155.8 to 60.2 nm in the first 20 h of cardiomyocyte culture. Furthermore, the cardiomyocytes cultured in long-term medium and short-term medium (ACCT) for 10 h exhibit distinct difference in adhesion rate as well as cell−substrate distance (72 vs 68 nm).

A number of techniques have been developed to monitor contractile function in isolated cardiac myocytes. While invaluable observations have been gained from these methodologies in understanding the contractile processes of the heart, they are invariably limited by their in vitro conditions. The present challenge is to develop innovative assays to mimic the in vivo milieu so as to allow a more physiological assessment of cardiac myocyte contractile forces. Here we demonstrate the use of a silicone elastomer, poly(dimethylsiloxane) (PDMS), to simultaneously orient adult cardiac myocytes in primary culture and measure the cellular forces in a three-dimensional substrate. The realignment of adult cardiac myocytes in long-term culture (7 days) was achieved due to directional reassembly of the myofibrils along the parallel polymeric sidewalls. The cellular mechanical forces were recorded in situ by observing the deformation of the micropillars embedded in the substrate. By coupling the cellular mechanical force measurements with on-chip cell orientation, this novel assay is expected to provide a means of a more physiological assessment of single cardiac myocyte contractile function and may facilitate the future development of in vitro assembled functional cardiac tissue.

Myofibrils are functional contractile elements in skeleton and cardiac muscle cells of vertebrates and in skeleton muscle cells of invertebrates. Each myofibril consists of parallel filaments, allowing maximal contraction performance. When these muscle cells are cultured in vitro, this parallel arrangement is often disrupted due to cell remodeling. The contractile performance of the muscle cells is thus deteriorated. In this paper, we present a microstructured polymeric substrate to regulate the reassembly of the myofibrils during cell remodeling. The results show the myofibrils in long-term cultured heart muscle cells are reassembled directionally, along longitudinal axis of the microstructured polymeric sidewalls. This directional arrangement is further validated by monitoring the mechanical performance of the living cells, using embedded polymeric microstructures. This work opens a door for orientation of subcellular elements using microstructured extracellular environments, and is the basis for development of in vitro assembled muscle patches.

The nanometer-scale in-plane deformation of a PDMS nanostructure array was demonstrated using a geometric moiré technique. The polymer nanostructures with cylindrical profile were fabricated by using the combination of e-beam lithography, reactive ion etching, and replica molding. The moiré pattern is generated by the inference between the polymer nanostructures and the scanning lines of a CCD video camera. A uniform thermal expansion was induced in the polymer nanostructures. The moiré pattern change was observed at different temperatures from 298 to 398 K. The change of the strain/pitch in the nanostructures as the temperature varied was calculated from the pure extension and the angular moiré fringes. The results show the feasibility of using such polymer nanostructures as force sensors to measure the mechanical forces on the order of a few nanonewtons or less. This work has a representative application in mechanics study of the biological objects, e.g. living cells and protein, under their environmental conditions.

This paper reports a PDMS microchip for subcellular mechanics study, which consists of micrometer scale polymeric pillars on a plain substrate for cellular force measurement. The chip reported here differs from those proposed in previous work in that a flexible polymer microfabrication technique was applied for manufacturing the polymeric microstructures with various aspect ratios. This allows for the measurement of a range of forces encountered at the subcellular level. The microchip was integrated with a perfusion chamber to allow for precise control of the extracellular environment, thus facilitating cellular mechanics studies under various physiological conditions. A biocompatibility test was carried out using two types of mammalian cells (fibroblasts and cardiac myocytes). The results show satisfactory abundances of both types of cells, with no adverse affect of the PDMS microchip on cell viability. A proof-of-concept force measurement was performed in single contracting cardiac myocytes. The force distribution with a subcellular resolution conforms to the physiologic behavior of cardiac myoctes, indicating the potential application of the reported microchip in subcellular mechanics study.

This paper reports a flexible fabrication process to manufacture polymeric microstructures with various aspect ratios using a micro molding process. In this study, a silicon template with deep holes (2 µm in diameter) was fabricated and a two-phase vacuum pressure-assisted process was conducted. The liquid polymer contacting with the template was raised into the deep holes due to the differential pressure between the trapped air in the holes and the ambient pressure. The microstructures were solidified by thermal curing at an elevated temperature (65 °C). The height of the resulting polymeric microstructures, ranging from submicron to more than 10 µm, was controlled by tuning the operation parameters in the micro molding process. A proof-of-principle experiment was carried out in isolated cardiac myocytes to measure the cellular forces towards the polymeric microstructures. Moreover, simultaneous cell alignment and force measurement was demonstrated using an advanced structure fabricated using such a technique. This approach reported here differs from the existing ones in that it enables the "1-to-n" replication, thereby lowering the fabrication cost and complexity. This study has a potential application in a variety of polymer-based micro total analysis systems, especially where the polymer microstructures serve as mechanical sensors.

This paper presents cellular mechanics study in isolated cardiac myocytes using a micro-molded polydimethylsiloxane (PDMS) pillars array. The PDMS pillars were fabricated using a specialized micro-molding process where template fabrication and polymer replication were minimized. The spring constant of the subject pillars was determined considering the enlarged root and scallop-like notches. The cellular mechanical force was derived from displacements of individual pillars upon multiplication with the locally determined spring constant. Experiments were conducted to achieve the subcellular force distribution within single cardiac myocyte. Furthermore, the force response of isolated cardiac myocytes upon an isoproterenol perfusion was in situ monitored. The measurements conform to the effect of applied chemical stimulus, suggesting that this approach has the physiology and pathophysiology potential to be a basis for subcellular mechanics study in cardiomyopathy field.

Polymeric material has been utilized as mechanical sensors to measure microscopic cellular forces. Since many polymers are not readily compatible with conventional lithography, fabrication of numerous molds is inevitably a part of the process, compromising low cost and process simplicity. In this letter, we apply a flexible fabrication process to manufacture polymeric mechanical sensors with various aspect ratios from a single rigid mold. A proof-of-principle measurement was carried out in isolated cardiac myocytes. The results conform to the physiologic behavior. This approach has the potential for evaluation of mechanical interaction between various biological units and the substrates while minimizing the fabrication cost and complexity.

Mechanical force is one of the most important parameters in cellular physiological behavior. To quantify the cellular force locally and more precisely, soft material probes, such as bulk polymeric surfaces or raised individual polymeric structures, have been developed which are deformable by the cell. The extent of deformation and the elastic properties of the probes allow for calculation of the mechanical forces exerted by the cell. Bulk polymeric surfaces have the disadvantage of requiring computational intensive calculations due to the continuous distortion of a large area, and investigators have attempted to address this problem by using raised polymeric structures to simplify the derivation of cellular mechanical force. These studies, however, have ignored the possibility of formation of local adhesions of the cell to the underlying base substrate, which could result in inaccurate cellular force measurements. Clearly, there is a need to develop polymeric structures that can efficiently isolate the cells from the underlying base substrate, in order to eliminate the continuous distortion problem. In this paper, we demonstrate the measurement of cellular force in isolated cardiac myocytes using single-spaced polymeric microstructures. Each structure is 2 µm in diameter and single-spaced packed. This geometry of the structures successfully isolates the cells from the underlying substrate. Displacement of the structures was measured in areas underneath the attached cell and at areas in close proximity to the cell. The results show that the individual structures underneath the cell were significantly displaced whereas no substantial strain in the underlying base substrate was detected. The mechanical force of the cell was derived from the displacements of individual structures upon multiplication with the locally determined spring constant. The force distribution reveals a parallel alignment as well as a periodic motion of the contractile units of the myocyte. The flexible fabrication methodology of the polymeric substrate and straightforward determination of minute forces provide a useful way to study cellular mechanical force.