Design of a 3D printed, motorized, uniaxial cell stretcher for microscopic and biochemical analysis of mechanotransduction.

Cells respond to mechanical cues from their environment through a process of mechanosensing and mechanotransduction. Cell stretching devices are important tools to study the molecular pathways responsible for cellular responses to mechanobiological processes. We describe the development and testing of a uniaxial cell stretcher that has applications for microscopic as well as biochemical analyses. By combining simple fabrication techniques with adjustable control parameters, the stretcher is designed to fit a variety of experimental needs. The stretcher can be used for static and cyclic stretching. As a proof of principle, we visualize stretch induced deformation of cell nuclei via incremental static stretch, and changes in IEX1 expression via cyclic stretching. This stretcher is easily modified to meet experimental needs, inexpensive to build, and should be readily accessible for most laboratories with access to 3D printing.

A portable pen-sized instrumentation to measure stiffness of soft tissues in vivo.

Quantitative assessment of soft tissue elasticity is crucial to a broad range of applications, such as biomechanical modeling, physiological monitoring, and tissue diseases diagnosing. However, the modulus measurement of soft tissues, particularly in vivo, has proved challenging since the instrument has to reach the site of soft tissue and be able to measure in a very short time. Here, we present a simple method to measure the elastic modulus of soft tissues on site by exploiting buckling of a long slender bar to quantify the applied force and a spherical indentation to extract the elastic modulus. The method is realized by developing a portable pen-sized instrument (EPen: Elastic modulus pen). The measurement accuracies are verified by independent modulus measures using commercial nanoindenter. Quantitative measurements of the elastic modulus of mouse pancreas, healthy and cancerous, surgically exposed but attached to the body further confirm the potential clinical utility of the EPen.

Biological physics by high-speed atomic force microscopy.

While many fields have contributed to biological physics, nanotechnology offers a new scale of observation. High-speed atomic force microscopy (HS-AFM) provides nanometre structural information and dynamics with subsecond resolution of biological systems. Moreover, HS-AFM allows us to measure piconewton forces within microseconds giving access to unexplored, fast biophysical processes. Thus, HS-AFM provides a tool to nourish biological physics through the observation of emergent physical phenomena in biological systems. In this review, we present an overview of the contribution of HS-AFM, both in imaging and force spectroscopy modes, to the field of biological physics. We focus on examples in which HS-AFM observations on membrane remodelling, molecular motors or the unfolding of proteins have stimulated the development of novel theories or the emergence of new concepts. We finally provide expected applications and developments of HS-AFM that we believe will continue contributing to our understanding of nature, by serving to the dialogue between biology and physics. This article is part of a discussion meeting issue 'Dynamic in situ microscopy relating structure and function'.

Mechano-Pharmacological Testing of L-Type Ca2+ Channel Modulators via Human Vascular Celldrum Model.

BACKGROUND/AIMS: This study aimed to establish a precise and well-defined working model, assessing pharmaceutical effects on vascular smooth muscle cell monolayer in-vitro. It describes various analysis techniques to determine the most suitable to measure the biomechanical impact of vasoactive agents by using CellDrum technology. METHODS: The so-called CellDrum technology was applied to analyse the biomechanical properties of confluent human aorta muscle cells (haSMC) in monolayer. The cell generated tensions deviations in the range of a few N/m² are evaluated by the CellDrum technology. This study focuses on the dilative and contractive effects of L-type Ca2+ channel agonists and antagonists, respectively. We analyzed the effects of Bay K8644, nifedipine and verapamil. Three different measurement modes were developed and applied to determine the most appropriate analysis technique for the study purpose. These three operation modes are called, particular time mode" (PTM), "long term mode" (LTM) and "real-time mode" (RTM). RESULTS: It was possible to quantify the biomechanical response of haSMCs to the addition of vasoactive agents using CellDrum technology. Due to the supplementation of 100nM Bay K8644, the tension increased approximately 10.6% from initial tension maximum, whereas, the treatment with nifedipine and verapamil caused a significant decrease in cellular tension: 10nM nifedipine decreased the biomechanical stress around 6,5% and 50nM verapamil by 2,8%, compared to the initial tension maximum. Additionally, all tested measurement modes provide similar results while focusing on different analysis parameters. CONCLUSION: The CellDrum technology allows highly sensitive biomechanical stress measurements of cultured haSMC monolayers. The mechanical stress responses evoked by the application of vasoactive calcium channel modulators were quantified functionally (N/m²). All tested operation modes resulted in equal findings, whereas each mode features operation-related data analysis.

Pneumatic unidirectional cell stretching device for mechanobiological studies of cardiomyocytes.

In this paper, we present a transparent mechanical stimulation device capable of uniaxial stimulation, which is compatible with standard bioanalytical methods used in cellular mechanobiology. We validate the functionality of the uniaxial stimulation system using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The pneumatically controlled device is fabricated from polydimethylsiloxane (PDMS) and provides uniaxial strain and superior optical performance compatible with standard inverted microscopy techniques used for bioanalytics (e.g., fluorescence microscopy and calcium imaging). Therefore, it allows for a continuous investigation of the cell state during stretching experiments. The paper introduces design and fabrication of the device, characterizes the mechanical performance of the device and demonstrates the compatibility with standard bioanalytical analysis tools. Imaging modalities, such as high-resolution live cell phase contrast imaging and video recordings, fluorescent imaging and calcium imaging are possible to perform in the device. Utilizing the different imaging modalities and proposed stretching device, we demonstrate the capability of the device for extensive further studies of hiPSC-CMs. We also demonstrate that sarcomere structures of hiPSC-CMs organize and orient perpendicular to uniaxial strain axis and thus express more maturated nature of cardiomyocytes.

A Comparison Between Emerging and Current Biophysical Methods for the Assessment of Higher-Order Structure of Biopharmaceuticals.

The higher-order structure (HOS) of protein therapeutics is a critical quality attribute directly related to their function. Traditionally, the HOS of protein therapeutics has been characterized by methods with low to medium structural resolution such as Fourier-transform infrared (FTIR), circular dichroism (CD), and intrinsic fluorescence spectroscopy, and differential scanning calorimetry (DSC). Recently, high-resolution nuclear magnetic resonance (NMR) methods have emerged as powerful tools for HOS characterization. NMR is a multi-attribute method with unique capabilities to provide information about all the structural levels of proteins in solution. We have in this study compared 1 D 1H Profile NMR with the established biophysical methods for HOS assessments using a set of blended samples of the monoclonal antibodies belonging to the subclasses IgG1 and IgG2. The study shows that Profile NMR can distinguish between most sample combinations (93%), DSC can differentiate 61% of the sample combinations, and near-ultraviolet CD spectroscopy can differentiate 52% of the sample combinations, whereas no significant distinction could be made between any samples using FTIR or intrinsic fluorescence. Our data therefore show that NMR has superior ability to address differences in HOS, a feature that could be directly applicable in comparability and similarity assessments.

Brillouin microscopy: an emerging tool for mechanobiology.

The role and importance of mechanical properties of cells and tissues in cellular function, development and disease has widely been acknowledged, however standard techniques currently used to assess them exhibit intrinsic limitations. Recently, Brillouin microscopy, a type of optical elastography, has emerged as a non-destructive, label- and contact-free method that can probe the viscoelastic properties of biological samples with diffraction-limited resolution in 3D. This led to increased attention amongst the biological and medical research communities, but it also sparked debates about the interpretation and relevance of the measured physical quantities. Here, we review this emerging technology by describing the underlying biophysical principles and discussing the interpretation of Brillouin spectra arising from heterogeneous biological matter. We further elaborate on the technique's limitations, as well as its potential for gaining insights in biology, in order to guide interested researchers from various fields.

Dynamic Bioreactors with Integrated Microfabricated Devices for Mechanobiological Screening.

Biomechanical stimulation is a common strategy to improve the growth, maturation, and function of a variety of engineered tissues. However, identifying optimized biomechanical conditioning protocols is challenging, as cell responses to mechanical stimuli are modulated by other multifactorial microenvironmental cues, including soluble factors and biomaterial properties. Traditional bioreactors lack the throughput necessary for combinatorial testing of cell activity in mechanically stimulated engineered tissues. Microfabricated systems can improve experimental throughput, but often do not provide uniform mechanical loading, are challenging to use, lack robustness, and offer limited amounts of cells and tissue for analysis. To address the need for higher-throughput, combinatorial testing of cell activity in a tissue engineering context, we developed a hybrid approach, in which flexible polydimethylsiloxane microfabricated inserts were designed to simultaneously generate multiple tensile strains when stretched cyclically in a standard dynamic bioreactor. In the embodiment presented in this study, each insert contained an array of 35 dog bone-shaped wells in which cell-seeded microscale hydrogels can be polymerized, with up to eight inserts stretched simultaneously in the bioreactor. Uniformity of the applied strains, both along the length of a microtissue and across multiple microtissues at the same strain level, was confirmed experimentally. In proof-of-principle experiments, the combinatorial effects of dynamic strain, biomaterial stiffness, and transforming growth factor (TGF)-ß1 stimulation on myofibroblast differentiation were tested, revealing both known and novel interaction effects and suggesting tissue engineering strategies to regulate myofibroblast activation. This platform is expected to have wide applicability in systematically probing combinations of mechanobiological tissue engineering parameters for desired effects on cell fate and tissue function. Impact Statement In this study, we introduce a dynamic bioreactor system incorporating microfabricated inserts to enable systematic probing of the effects of combinations of mechanobiological parameters on engineered tissues. This novel platform offers the ease of use, robustness, and well-defined mechanical strain stimuli inherent in traditional dynamic bioreactors, but significantly improves throughput (up to 280 microtissues can be tested simultaneously in the embodiment presented in this study). This platform has wide applicability to systematically probe combinations of dynamic mechanical strain, biomaterial properties, biochemical stimulation, and other parameters for desired effects on cell fate and engineered tissue development.

Application of 3D Printing Technology for Design and Manufacturing of Customized Components for a Mechanical Stretching Bioreactor.

Three-dimensional (3D) printing represents a key technology for rapid prototyping, allowing easy, rapid, and low-cost fabrication. In this work, 3D printing was applied for the in-house production of customized components of a mechanical stretching bioreactor with potential application for cardiac tissue engineering and mechanobiology studies. The culture chamber housing and the motor housing were developed as functional permanent parts, aimed at fixing the culture chamber position and at guaranteeing motor watertightness, respectively. Innovative sample holder prototypes were specifically designed and 3D-printed for holding thin and soft biological samples during cyclic stretch culture. The manufactured components were tested in-house and in a cell biology laboratory. Moreover, tensile tests and finite element analysis were performed to investigate the gripping performance of the sample holder prototypes. All the components showed suitable performances in terms of design, ease of use, and functionality. Based on 3D printing, the bioreactor optimization was completely performed in-house, from design to fabrication, enabling customization freedom, strict design-to-prototype timing, and cost and time effective testing, finally boosting the bioreactor development process.

Feasibility of using a fully immersive virtual reality system for kinematic data collection.

Commercially-available Virtual Reality (VR) systems have the potential to be effective tools for simultaneous visual manipulation and kinematic data collection. Previously, these systems have been integrated with research-grade motion capture systems to provide both functionalities; however, they are yet to be used as stand-alone systems for kinematic data collection. The present study aimed to validate the HTC VIVE VR system for kinematic data collection by evaluating the accuracy of its position and orientation signals. The VIVE controller and tracker were each compared to a Polhemus Liberty magnetic tracking system sensor for angular and translational measurement error and signal drift. A sensor from each system was mounted to opposite ends of a rigid segment which was driven through fifty rotations and fifty translations. Mean angular errors for both the VIVE tracker and controller were below 0.4°. Mean translational error for both sensors was below 3 mm. Drift in the Liberty signal components was consistently lower than drift in VIVE components. However, all mean rotational drift measures were below 0.1° and all mean translational measures were below 0.35 mm. These data indicate that the HTC VIVE system has the potential to be a valid and reliable means of kinematic data collection. However, further investigation is necessary to determine the VIVE's suitability for capturing extremely minute or high-volume movements.

Comparison of posturographic outcomes between two different devices.

The Interactive Balance System (IBS), a posturography device for assessing posture control, is widely used in clinical and rehabilitation settings. However, data on the validity of the device are unavailable. Fluctuations of the center of pressure (COP) were measured in 24 healthy participants (age: 29 ±â€¯5 (mean ±â€¯SD) years, 12 females) synchronously using the IBS, which was rigidly mounted on a Kistler platform. Four different bipedal conditions were examined: eyes open or closed on stable or soft surfaces. Time series were compared using congruity (CON, proportion of the measurement time during which values of both devices changed similarly in direction), whereas IBS-specific postural outcomes were correlated with traditional postural control outcomes of the Kistler force platform. The time-displacement curves showed similar shapes for CON (>0.9) for each of the four standing conditions without differences between male and female participants (P > 0.39). The path length results of both devices showed very high linear associations, explaining on average 92% (medio-lateral) or 96% (anterior-posterior) of the common variance. The Kistler path length of the anterior-posterior direction revealed nearly perfect linear associations with the stability index of the IBS (r2 > 0.99). The results of this study indicate that the IBS provides valid posturographic results. Since the medial-lateral and anterior-posterior trajectories of the IBS can be used to calculate COP fluctuations, comparisons between different measurement systems are possible.

Foot centre of pressure and ground reaction force during quadriceps resistance exercises; a comparison between force plates and a pressure insole system.

The study compared the centre of pressure measurements (COP) and vertical ground reaction forces (vGRF) from a pressure insole system to that from force plates (FP) during two flywheel quadriceps resistance exercises: leg press and squat. The comparison was performed using a motion capture system and simultaneous measurements of COP and vGRF from FP and insoles. At lower insole-vGRF (<250 N/insole) COP accuracy deteriorated and those data were excluded from further analysis. The insoles systematically displaced the COP slightly posteriorly and medially compared to the FP measurements. Pearson's coefficient of correlation (r) between insole- and FP-COP showed good agreement in both the anteroposterior (squat: r = 0.96, leg press: r = 0.97) and mediolateral direction (squat: r = 0.84, leg press: r = 0.90), whereas the root-mean-square errors (RMSE) were lower in the mediolateral (squat: 3.9 mm, leg press: 4.5 mm) than the anteroposterior (squat and leg press: 11.8 mm) direction. Vertical GRF was slightly overestimated by the insoles in leg press and RMSE were greater in leg press (8% of peak force) than in squat (6%). Overall, results were within the range of previous studies performed on gait. The strong agreement between insole and FP measurements indicates that insoles may replace FPs in field applications and biomechanical computations during resistance exercise, provided that the applied force is sufficient.

Head mounted displays for capturing head kinematics in postural tasks.

Tracking head motion in a simple, portable and accurate manner during performance of postural tasks in a virtual reality environment could have important implications for investigating normal and pathological head kinematics. We investigated concurrent validity of head tracking of two Head Mounted Displays (HMDs), Oculus Rift and HTC Vive, vs. a gold-standard motion capture system (Qualisys). Head kinematics of N = 20 healthy young adults was quantified during static and dynamic postural tasks. While wearing the Oculus Rift or HTC Vive, participants observed moving stars (static tasks) or a flying ball (dynamic task). Head kinematics were recorded simultaneously by the Rift or Vive and Qualisys camera system. We calculated head directional path, acceleration in 6 directions and volume of translation movement. Intra-Class Correlations (ICC) and 95% Limits of agreement were calculated. Most ICC values were around 0.9 with several at 0.99 indicating excellent agreement between the HMDs and Qualisys. Weaker agreement was observed for vertical displacement during a static task and moderate agreement was observed pitch and yaw displacement during a dynamic task. A negative bias of a small magnitude (indicating more movement in VR) was observed for most variables in static tasks, while a positive bias was observed for most variables in the dynamic task (indicating less movement in VR). Our results generally support the concurrent validity of Oculus Rift and HTC Vive head tracking during static and dynamic standing tasks in healthy young adults. Specific task- and direction-dependent differences should be considered when planning measurement studies using these novel tools.

New considerations for collecting biomechanical data using wearable sensors: Number of level runs to define a stable running pattern with a single IMU.

Wearable technology can be used to quantify running biomechanical patterns in a runner's natural environment, however, changes in external factors during outdoor running may influence a runner's typical gait pattern. Therefore, the purpose of this study was to determine how many runs are needed to define a stable or typical running pattern. Six biomechanical variables were recorded using a single wearable sensor placed on the lower back during ten outdoor runs for twelve runners. Univariate and multivariate distributions were created and based on the probability density function, the percent of similar data points (within 95%) from each unique run for the same runner were determined. Stability was defined when the addition of data from a new run resulted in less than a 5% change in the probability density function. To cross-validate, the percent of similar data points at stability was compared between the same and different runners using a repeated-measures MANOVA (Bonferroni-corrected α = 0.007). The maximum number of runs needed to reach stability for univariate and multivariate analyses was four and five, respectively. There was a significant overall effect on similar data points between the same and different runners (p = 0.001), with a greater percent of similar data points for the same runner compared to other runners (p < 0.007). Based on biomechanical data collected using a single wearable sensor placed on the lower back, this is the first study to show that four (univariate) to five (multivariate) runs are needed to establish a stable running pattern in real-world settings.

Validity of a novel method to measure vertical oscillation during running using a depth camera.

Recent advancements in low-cost depth cameras may provide a clinically accessible alternative to conventional three-dimensional (3D) multi-camera motion capture systems for gait analysis. However, there remains a lack of information on the validity of clinically relevant running gait parameters such as vertical oscillation (VO). The purpose of this study was to assess the validity of measures of VO during running gait using raw depth data, in comparison to a 3D multi-camera motion capture system. Sixteen healthy adults ran on a treadmill at a standard speed of 2.7 m/s. The VO of their running gait was simultaneously collected from raw depth data (Microsoft Kinect v2) and 3D marker data (Vicon multi-camera motion capture system). The agreement between the VO measures obtained from the two systems was assessed using a Bland-Altman plot with 95% limits of agreement (LOA), a Pearson's correlation coefficient (r), and a Lin's concordance correlation coefficient (rc). The depth data from the Kinect v2 demonstrated excellent results across all measures of validity (r = 0.97; rc = 0.97; 95% LOA = -8.0 mm - 8.7 mm), with an average absolute error and percent error of 3.7 (2.1) mm and 4.0 (2.0)%, respectively. The findings of this study have demonstrated the ability of a low cost depth camera and a novel tracking method to accurately measure VO in running gait.

Studying Cell Mechanobiology in 3D: The Two-Photon Lithography Approach.

Two-photon lithography is a laser writing technique that can produce 3D microstructures with resolutions below the diffraction limit. This review focuses on its applications to study mechanical properties of cells, an emerging field known as mechanobiology. We review 3D structural designs and materials in the context of new experimental designs, including estimating forces exerted by single cells, studying selective adhesion on substrates, and creating 3D networks of cells. We then focus on emerging applications, including structures for assessing cancer cell invasiveness, whose migration properties depend on the cell mechanical response to the environment, and 3D architectures and materials to study stem cell differentiation, as 3D structure shape and patterning play a key role in defining cell fates.

Mapping heterogeneity of cellular mechanics by multi-harmonic atomic force microscopy.

The goal of mechanobiology is to understand the links between changes in the physical properties of living cells and normal physiology and disease. This requires mechanical measurements that have appropriate spatial and temporal resolution within a single cell. Conventional atomic force microscopy (AFM) methods that acquire force curves pointwise are used to map the heterogeneous mechanical properties of cells. However, the resulting map acquisition time is much longer than that required to study many dynamic cellular processes. Dynamic AFM (dAFM) methods using resonant microcantilevers are compatible with higher-speed, high-resolution scanning; however, they do not directly acquire force curves and they require the conversion of a limited number of instrument observables to local mechanical property maps. We have recently developed a technique that allows commercial AFM systems equipped with direct cantilever excitation to quantitatively map the viscoelastic properties of live cells. The properties can be obtained at several widely spaced frequencies with nanometer-range spatial resolution and with fast image acquisition times (tens of seconds). Here, we describe detailed procedures for quantitative mapping, including sample preparation, AFM calibration, and data analysis. The protocol can be applied to different biological samples, including cells and viruses. The transition from dAFM imaging to quantitative mapping should be easily achievable for experienced AFM users, who will be able to set up the protocol in <30 min.

Tank Circuit for Ultrafast Single-Particle Detection in Micropores.

We present an ultrafast single submicron particle detection method based on a half-bowtie coplanar waveguide. The method is capable of resolving the translocation of these particles at a bandwidth greater than 30 MHz. We compare experimentally the simultaneous use of our radio-frequency technique with conventional dc based resistive pulse recordings and find that our method has a throughput that is enhanced by 2 orders of magnitude. The technique incorporates a microfluidic circuit and has the potential to be employed for screening microparticles and biological cells at frequencies in excess of 1 GHz.

Rational Design of Semiconductor Nanostructures for Functional Subcellular Interfaces.

One of the fundamental questions guiding research in the biological sciences is how cellular systems process complex physical and environmental cues and communicate with each other across multiple length scales. Importantly, aberrant signal processing in these systems can lead to diseases that can have devastating impacts on human lives. Biophysical studies in the past several decades have demonstrated that cells can respond to not only biochemical cues but also mechanical and electrical ones. Thus, the development of new materials that can both sense and modulate all of these pathways is necessary. Semiconducting nanostructures are an emerging class of discovery platforms and tools that can push the limits of our ability to modulate and sense biological behaviors for both fundamental research and clinical applications. These materials are of particular interest for interfacing with cellular systems due to their matched dimension with subcellular components (e.g., cytoskeletal filaments), and easily tunable properties in the electrical, optical and mechanical regimes. Rational design via traditional or new approaches, such as nanocasting and mesoscale chemical lithography, can allow us to control micro- and nanoscale features in nanowires to achieve new biointerfaces. Both processes endogenous to the target cell and properties of the material surface dictate the character of these interfaces. In this Account, we focus on (1) approaches for the rational design of semiconducting nanowires that exhibit unique structures for biointerfaces, (2) recent fundamental discoveries that yield robust biointerfaces at the subcellular level, (3) intracellular electrical and mechanical sensing, and (4) modulation of cellular behaviors through material topography and remote physical stimuli. In the first section, we discuss new approaches for the synthetic control of micro- and nanoscale features of these materials. In the second section, we focus on achieving biointerfaces with these rationally designed materials either intra- or extracellularly. We last delve into the use of these materials in sensing mechanical forces and electrical signals in various cellular systems as well as in instructing cellular behaviors. Future research in this area may shift the paradigm in fundamental biophysical research and biomedical applications through (1) the design and synthesis of new semiconductor-based materials and devices that interact specifically with targeted cells, (2) the clarification of many developmental, physiological, and anatomical aspects of cellular communications, (3) an understanding of how signaling between cells regulates synaptic development (e.g., information like this would offer new insight into how the nervous system works and provide new targets for the treatment of neurological diseases), (4) and the creation of new cellular materials that have the potential to open up completely new areas of application, such as in hybrid information processing systems.