Cell Stretcher Research Articles

Pneumatic Non-Equibiaxial Cell Stretching Device With Live-Cell Imaging.

Adherent cell behavior is influenced by a complex interplay of factors, including chemical and mechanical signals. In vitro experiments that mimic the mechanical environment experienced by cells in vivo are crucial for understanding cellular behavior and the progression of disease. In this study, we developed and validated a low-cost pneumatically-controlled cell stretcher with independent control of strain in two directions of a membrane, enabling unequal biaxial stretching and real-time microscopy during actuation. The stretching was achieved by two independent pneumatic channels controlled by electrical signals. We used finite element simulations to compute the membrane's strain field and particle tracking algorithms based on image processing techniques to validate the strain fields and measure the cell orientation and morphology. The device can supply uniaxial, equibiaxial, and unequal biaxial stretching up to [Formula: see text] strain in each direction at a frequency of [Formula: see text], with a strain measurement error of less than 1%. Through live cell imaging, we determined that distinct stretching patterns elicited differing responses and alterations in cell orientation and morphology, particularly in terms of cell length and area. The device successfully provides a large, uniform, and variable strain field for cell experiments, while also enabling real-time, live cell imaging. This scalable, low-cost platform provides mechanical stimulation to cell cultures by independently controlling strains in two directions. This could contribute to a deeper understanding of cellular response to bio-realistic strains and could be useful for future in vitro drug testing platforms.

Cell Stretcher Assay to Analyze Mechanoresponses to Cyclic Stretching.

In their natural environment, most cells and tissues are continuously exposed to cyclic mechanical strain. Sensing these stimuli by mechanosensory proteins and subsequent conversion into a variety of biological responses (referred to as mechanotransduction) are key processes for tissue homeostasis, survival, and differentiation. Perturbations of underlying signaling pathways lead to severe diseases in vivo (Urciuoli E, Peruzzi B, Int J Mol Sci 21(24). https://doi.org/10.3390/ijms21249426, (2020)). In addition, cellular mechanoresponses to cyclic stretching of an isolated single cell differ from those of a cell monolayer, network, or even three-dimensional tissue. Since these processes depend on various physical and biological parameters, the development of a precise, well-characterized, and highly reproducible but also easily tunable stretcher assay is indispensable. Here, we describe the fabrication of defined elastic substrates and their application in cyclic stretching of cultured cells in a custom-made cell stretcher device. We focus on the detailed description of the system and provide a possibility for mechanoresponse characterization, using the analysis of actin stress fiber orientation as exemplary mechanoresponse to cyclic stretching of adherent cells.

Supracellular measurement of spatially varying mechanical heterogeneities in live monolayers

The mechanical properties of tissues have profound impacts on a wide range of biological processes such as embryo development (1,2), wound healing (3–6), and disease progression (7). Specifically, the spatially varying moduli of cells largely influence the local tissue deformation and intercellular interaction. Despite the importance of characterizing such a heterogeneous mechanical property, it has remained difficult to measure the supracellular modulus field in live cell layers with a high-throughput and minimal perturbation. In this work, we developed a monolayer effective modulus measurement by integrating a custom cell stretcher, light microscopy, and AI-based inference. Our approach first quantifies the heterogeneous deformation of a slightly stretched cell layer and converts the measured strain fields into an effective modulus field using an AI inference. This method allowed us to directly visualize the effective modulus distribution of thousands of cells virtually instantly. We characterized the mean value, SD, and correlation length of the effective cell modulus for epithelial cells and fibroblasts, which are in agreement with previous results. We also observed a mild correlation between cell area and stiffness in jammed epithelia, suggesting the influence of cell modulus on packing. Overall, our reported experimental platform provides a valuable alternative cell mechanics measurement tool that can be integrated with microscopy-based characterizations.

An Easy-to-Fabricate Cell Stretcher Reveals Density-Dependent Mechanical Regulation of Collective Cell Movements in Epithelia

IntroductionMechanical forces regulate many facets of cell and tissue biology. Studying the effects of forces on cells requires real-time observations of single- and multi-cell dynamics in tissue models during controlled external mechanical input. Many of the existing devices used to conduct these studies are costly and complicated to fabricate, which reduces the availability of these devices to many laboratories.MethodsWe show how to fabricate a simple, low-cost, uniaxial stretching device, with readily available materials and instruments that is compatible with high-resolution time-lapse microscopy of adherent cell monolayers. In addition, we show how to construct a pressure controller that induces a repeatable degree of stretch in monolayers, as well as a custom MATLAB code to quantify individual cell strains.ResultsAs an application note using this device, we show that uniaxial stretch slows down cellular movements in a mammalian epithelial monolayer in a cell density-dependent manner. We demonstrate that the effect on cell movement involves the relocalization of myosin downstream of Rho-associated protein kinase (ROCK).ConclusionsThis mechanical device provides a platform for broader involvement of engineers and biologists in this important area of cell and tissue biology. We used this device to demonstrate the mechanical regulation of collective cell movements in epithelia.

AFM and Fluorescence Microscopy of Single Cells with Simultaneous Mechanical Stimulation via Electrically Stretchable Substrates.

We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic deformations to adherent cells in culture, (ii) allows the visualization of cells under fluorescence microscopy, and (iii) allows atomic force microscopy nanoindentation measurements of the mechanical properties of the cells. The cell stretcher device relies on a dielectric elastomer film that can be electro-actuated and acts as the cell culture substrate. The shape and position of the electrodes actuating the film can be controlled by design in order to obtain specific deformations across the cell culture chamber. By using optical markers we characterized the strain fields under different electrode configurations and applied potentials. The combined setup, which includes the cell stretcher device, an atomic force microscope, and an inverted optical microscope, can assess in situ and with sub-micron spatial resolution single cell topography and elasticity, as well as ion fluxes, during the application of static deformations. Proof of performance on fibroblasts shows a reproducible increase in the average cell elastic modulus as a response to applied uniaxial stretch of just 4%. Additionally, high resolution topography and elasticity maps on a single fibroblast can be acquired while the cell is deformed, providing evidence of long-term instrumental stability. This study provides a proof-of-concept of a novel platform that allows in situ and real time investigation of single cell mechano-transduction phenomena with sub-cellular spatial resolution.

A Fully Integrated Arduino-Based System for the Application of Stretching Stimuli to Living Cells and Their Time-Lapse Observation: A Do-It-Yourself Biology Approach

Mechanobiology has nowadays acquired the status of a topic of fundamental importance in a degree in Biological Sciences. It is inherently a multidisciplinary topic where biology, physics and engineering competences are required. A course in mechanobiology should include lab experiences where students can appreciate how mechanical stimuli from outside affect living cell behaviour. Here we describe all the steps to build a cell stretcher inside an on-stage cell incubator. This device allows exposing living cells to a periodic mechanical stimulus similar to what happens in physiological conditions such as, for example, in the vascular system or in the lungs. The reaction of the cells to the periodic mechanical stretching represents a prototype of a mechanobiological signal integrated by living cells. We also provide the theoretical and experimental aspects related to the calibration of the stretcher apparatus at a level accessible to researchers not used to dealing with topics like continuum mechanics and analysis of deformations. We tested our device by stretching cells of two different lines, U87-MG and Balb-3T3 cells, and we analysed and discussed the effect of the periodic stimulus on both cell reorientation and migration. We also discuss the basic aspects related to the quantitative analysis of the reorientation process and of cell migration. We think that the device we propose can be easily reproduced at low-cost within a project-oriented course in the fields of biology, biotechnology and medical engineering.

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

ABSTRACTCells 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 low-cost uniaxial cell stretcher for six parallel wells

Cells in the lungs, the heart, and numerous other organs, are constantly exposed to dynamic forces and deformations. To mimic these dynamic mechanical loading conditions and to study the resulting cellular responses such as morphological changes or the activation of biochemical signaling pathways, cells are typically seeded on flexible 2D substrates that are uniaxially or biaxially stretched. Here, we present an open-source cell stretcher built from parts of an Anet A8 3D printer. The cell stretcher is controlled by a fully programmable open-source software using GCode and Python. Up to six flexible optically clear substrates can be stretched simultaneously, allowing for comparative multi-batch biological studies including microscopic image analysis. The cell yield from the cell culture area of 4 cm2 per substrate is sufficient for Western-blot protein analysis. As a proof-of-concept, we study the activation of the Yes-associated protein (YAP) mechanotransduction pathway in response to increased cytoskeletal tension induced by uniaxial stretching of epithelial cells. Our data support the previously observed activation of the YAP transcription pathway by stretch-induced increase in cytoskeletal tension and demonstrate the suitability of the cell stretcher to study complex mechano-biological processes.

Effect of PAK Inhibition on Cell Mechanics Depends on Rac1.

Besides biochemical and molecular regulation, the migration and invasion of cells is controlled by the environmental mechanics and cellular mechanics. Hence, the mechanical phenotype of cells, such as fibroblasts, seems to be crucial for the migratory capacity in confined 3D extracellular matrices. Recently, we have shown that the migratory and invasive capacity of mouse embryonic fibroblasts depends on the expression of the Rho-GTPase Rac1, similarly it has been demonstrated that the Rho-GTPase Cdc42 affects cell motility. The p21-activated kinase (PAK) is an effector down-stream target of both Rho-GTPases Rac1 and Cdc42, and it can activate via the LIM kinase-1 its down-stream target cofilin and subsequently support the cell migration and invasion through the polymerization of actin filaments. Since Rac1 deficient cells become mechanically softer than controls, we investigated the effect of group I PAKs and PAK1 inhibition on cell mechanics in the presence and absence of Rac1. Therefore, we determined whether mouse embryonic fibroblasts, in which Rac1 was knocked-out, and control cells, displayed cell mechanical alterations after treatment with group I PAKs or PAK1 inhibitors using a magnetic tweezer (adhesive cell state) and an optical cell stretcher (non-adhesive cell state). In fact, we found that group I PAKs and Pak1 inhibition decreased the stiffness and the Young’s modulus of fibroblasts in the presence of Rac1 independent of their adhesive state. However, in the absence of Rac1 the effect was abolished in the adhesive cell state for both inhibitors and in their non-adhesive state, the effect was abolished for the FRAX597 inhibitor, but not for the IPA3 inhibitor. The migration and invasion were additionally reduced by both PAK inhibitors in the presence of Rac1. In the absence of Rac1, only FRAX597 inhibitor reduced their invasiveness, whereas IPA3 had no effect. These findings indicate that group I PAKs and PAK1 inhibition is solely possible in the presence of Rac1 highlighting Rac1/PAK I (PAK1, 2, and 3) as major players in cell mechanics.

An optofluidic "tweeze-and-drag" cell stretcher in a microfluidic channel.

The mechanical properties of biological cells are utilized as an inherent, label-free biomarker to indicate physiological and pathological changes of cells. Although various optical and microfluidic techniques have been developed for cell mechanical characterization, there is still a strong demand for non-contact and continuous methods. Here, by combining optical and microfluidic techniques in a single desktop platform, we demonstrate an optofluidic cell stretcher based on a "tweeze-and-drag" mechanism using a periodically chopped, tightly focused laser beam as an optical tweezer to trap a cell temporarily and a flow-induced drag force to stretch the cell in a microfluidic channel transverse to the tweezer. Our method leverages the advantages of non-contact optical forces and a microfluidic flow for both cell stretching and continuous cell delivery. We demonstrate the stretcher for mechanical characterization of rabbit red blood cells (RBCs), with a throughput of ∼1 cell per s at a flow rate of 2.5 μl h-1 at a continuous-wave laser power of ∼25 mW at a wavelength of 1064 nm (chopped at 2 Hz). We estimate the spring constant of RBCs to be ∼14.9 μN m-1. Using the stretcher, we distinguish healthy RBCs and RBCs treated with glutaraldehyde at concentrations of 5 × 10-4% to 2.5 × 10-3%, with a strain-to-concentration sensitivity of ∼-1529. By increasing the optical power to ∼45 mW, we demonstrate cell-stretching under a higher flow rate of 4 μl h-1, with a higher throughput of ∼1.5 cells per s and a higher sensitivity of ∼-2457. Our technique shows promise for applications in the fields of healthcare monitoring and biomechanical studies.

Microscale cell stretcher to generate spatially uniform equi-biaxial strain using an elastomeric membrane with a contoured thickness profile

Cellular response to mechanical signals is an intriguing research topic that is central to numerous fundamental questions in cell biology and has attracted extraordinary research interests. Delivering in-plane equi-biaxial strain towards adherent cells is a critical step for studying cellular mechanical responses. Mechanical straining is often performed by pushing a circular elastomeric membrane against a post or an O-ring or applying in-plane strain via through perforations on the membrane. Such methods, however, are not well compatible with miniaturization because the difficulties of device assembly and operation increase dramatically as the dimension of the membrane reduces. In this paper, we report a post-less elastomeric membrane with a contoured thickness profile for delivering in-plane equi-biaxial strain with significantly improved spatial uniformity. The easy assembly and operation allow for miniaturization of individual cellular mechanical stimulation sites to support parallel operation. The improvement of the spatial uniformity of the in-plane strain was experimentally validated with different membrane deformations. Cell reorientation and reorganization of actin filaments in fibroblasts under dynamic loading were examined using such elastomeric membranes. This work is expected to hasten the next-generation high throughput cellular mechanical stimulators for a broad range of cellular mechanobiological studies.

An ultra-fast mechanically active cell culture substrate

We present a mechanically active cell culture substrate that produces complex strain patterns and generates extremely high strain rates. The transparent miniaturized cell stretcher is compatible with live cell microscopy and provides a very compact and portable alternative to other systems. A cell monolayer is cultured on a dielectric elastomer actuator (DEA) made of a 30 μm thick silicone membrane sandwiched between stretchable electrodes. A potential difference of several kV’s is applied across the electrodes to generate electrostatic forces and induce mechanical deformation of the silicone membrane. The DEA cell stretcher we present here applies up to 38% tensile and 12% compressive strain, while allowing real-time live cell imaging. It reaches the set strain in well under 1 ms and generates strain rates as high as 870 s−1, or 87%/ms. With the unique capability to stretch and compress cells, our ultra-fast device can reproduce the rich mechanical environment experienced by cells in normal physiological conditions, as well as in extreme conditions such as blunt force trauma. This new tool will help solving lingering questions in the field of mechanobiology, including the strain-rate dependence of axonal injury and the role of mechanics in actin stress fiber kinetics.

A Low-Cost Mechanical Stretching Device for Uniaxial Strain of Cells: A Platform for Pedagogy in Mechanobiology.

Mechanical cues including stretch, compression, and shear stress play a critical role in regulating the behavior of many cell types, particularly those that experience substantial mechanical stress within tissues. Devices that impart mechanical stimulation to cells in vitro have been instrumental in helping to develop a better understanding of how cells respond to mechanical forces. However, these devices often have constraints, such as cost and limited functional capabilities, that restrict their use in research or educational environments. Here, we describe a low-cost method to fabricate a uniaxial cell stretcher that would enable widespread use and facilitate engineering design and mechanobiology education for undergraduate students. The device is capable of producing consistent and reliable strain profiles through the use of a servomotor, gear, and gear rack system. The servomotor can be programmed to output various waveforms at specific frequencies and stretch amplitudes by controlling the degree of rotation, speed, and acceleration of the servogear. In addition, the stretchable membranes are easy to fabricate and can be customized, allowing for greater flexibility in culture well size. We used the custom-built stretching device to uniaxially strain macrophages and cardiomyocytes, and found that both cell types displayed functional and cell shape changes that were consistent with the previous studies using commercially available systems. Overall, this uniaxial cell stretcher provides a more cost-effective alternative to study the effects of mechanical stretch on cells, and can therefore, be widely used in research and educational environments to broaden the study and pedagogy of cell mechanobiology.

Inertial Microfluidic Cell Stretcher (iMCS): Fully Automated, High-Throughput, and Near Real-Time Cell Mechanotyping.

Mechanical biomarkers associated with cytoskeletal structures have been reported as powerful label-free cell state identifiers. In order to measure cell mechanical properties, traditional biophysical (e.g., atomic force microscopy, micropipette aspiration, optical stretchers) and microfluidic approaches were mainly employed; however, they critically suffer from low-throughput, low-sensitivity, and/or time-consuming and labor-intensive processes, not allowing techniques to be practically used for cell biology research applications. Here, a novel inertial microfluidic cell stretcher (iMCS) capable of characterizing large populations of single-cell deformability near real-time is presented. The platform inertially controls cell positions in microchannels and deforms cells upon collision at a T-junction with large strain. The cell elongation motions are recorded, and thousands of cell deformability information is visualized near real-time similar to traditional flow cytometry. With a full automation, the entire cell mechanotyping process runs without any human intervention, realizing a user friendly and robust operation. Through iMCS, distinct cell stiffness changes in breast cancer progression and epithelial mesenchymal transition are reported, and the use of the platform for rapid cancer drug discovery is shown as well. The platform returns large populations of single-cell quantitative mechanical properties (e.g., shear modulus) on-the-fly with high statistical significances, enabling actual usages in clinical and biophysical studies.

Dynamic Imaging Reveals Coordinate Effects of Cyclic Stretch and Substrate Stiffness on Endothelial Integrity.

We describe an equibiaxial cell stretcher and hybrid, elastic membrane platform designed for dynamic imaging of cells on substrates with physiological stiffness undergoing cyclic stretch. Studies enabled by this device revealed that both substrate stiffness and cyclic stretch coordinately protect pulmonary endothelial monolayers against thrombin-induced disruption. The fluorescence imaging possible with the designed hybrid membranes further revealed similarities and differences in actin and cell dynamics during monolayer recovery. The improved live-cell imaging capabilities of this platform, when used in conjunction with fluorescent probes, will have broad applications for investigations of the impact of biochemical stimuli and mechanotransduction mechanisms on mechanically perturbed tissues.

Development of an Arbitrary Waveform Membrane Stretcher for Dynamic Cell Culture

In this paper, a novel cell stretcher design that mimics the real-time stretch of the heart wall is introduced. By culturing cells under stretched conditions that mimics the mechanical aspects of the native cardiac environment, better understanding on the role of biomechanical signaling on cell development can be achieved. The device utilizes a moving magnet linear actuator controlled through pulse-width modulated power combined with an automated closed loop feedback system for accurate generation of a designated mechanical stretch profile. The system's capability to stretch a cell culture membrane and accuracy of the designated frequency and waveform production for cyclic stretching were evaluated. Temperature and degradation assessments as well as a scalable design demonstrated the system's cell culture application for long term, in vitro studies.

A Uniaxial Cell Stretcher In Vitro Model Simulating Tissue Expansion of Plastic Surgery

Both in vivo and in vitro, cells are subjected to mechanical stress, which can affect their proliferation and differentiation. However, the detailed mechanisms of the accompanying morphologic and biochemical changes remain elusive. This is partially caused by the lack of suitable stretch model and method that mimic soft tissue expansion used in plastic surgery. In this study, we report the development of a novel model for uniaxial stretch cell mimicking tissue expansion. Fibroblasts, grown in plastic culture caskets, were stretched in a progressive and accumulative manner lasting from 1 to 4 days. Cell proliferation was measured using the methyl thiazolyl tetrazolium assay and the Western blot analysis of cell cycle proteins. Following the stretch protocol, fibroblasts elongated and aligned parallel to each other and to the applied strain vectors. Furthermore, stretch-stimulated cell proliferation was compared with the unstretched conditions. These results demonstrate that this uniaxial cell stretcher device and the accumulative stretch method are a suitable system for mimicking the tissue expansion process used in clinical applications. Not gradable.

Analysis of multiple physical parameters for mechanical phenotyping of living cells

Since the cytoskeleton is known to regulate many cell functions, an increasing amount of effort to characterize cells by their mechanical properties has occured. Despite the structural complexity and dynamics of the multicomponent cytoskeleton, mechanical measurements on single cells are often fit to simple models with two to three parameters, and those parameters are recorded and reported. However, different simple models are likely needed to capture the distinct mechanical cell states, and additional parameters may be needed to capture the ability of cells to actively deform. Our new approach is to capture a much larger set of possibly redundant parameters from cells' mechanical measurement using multiple rheological models as well as dynamic deformation and image data. Principal component analysis and network-based approaches are used to group parameters to reduce redundancies and develop robust biomechanical phenotyping. Network representation of parameters allows for visual exploration of cells' complex mechanical system, and highlights unexpected connections between parameters. To demonstrate that our biomechanical phenotyping approach can detect subtle mechanical differences, we used a Microfluidic Optical Cell Stretcher to mechanically stretch circulating human breast tumor cells bearing genetically-engineered alterations in c-src tyrosine kinase activation, which is known to influence reattachment and invasion during metastasis.

High-throughput cell stretcher

High-throughput cell stretcher

An array of 100 μm × 100 μm dielectric elastomer actuators with 80% strain for tissue engineering applications

Biological cells modulate their behavior, express genes, proliferate or differentiate in response to mechanical strains ranging from 1% to 20%. There currently exists no technique to apply strain to many targeted individual cells in a larger culture in order to perform parallelized high throughput testing. Dielectric elastomer actuators (DEAs) are compliant devices capable of generating large percentage strains with sub-second response time, ideally suited by their compliance for cell manipulation. We report an array of 100μm×100μm DEAs reaching up to 80% in-plane strain at an electric field of 240V/μm. The miniaturized DEAs are made by patterning 100μm wide compliant ion-implanted gold electrodes on both sides of a 30μm thick polydimethylsiloxane (PDMS) membrane. We report the important effect of uniaxial prestretch of the membrane on the microactuators’ performance; the largest strain is achieved by prestretching uniaxially by 175%. Each actuator is intended to have a single cell adhered to it in order to periodically stretch the cells to study the effect of mechanical stimulation on its biochemical responses. To avoid short-circuiting all the top electrodes by the conductive saline cell growth medium, a 20μm thick biocompatible PDMS layer is bonded on the actuators. In this configuration, 37% strain is achieved at 3.6kV with sub-second response. This device can be used as a high throughput single cell stretcher to apply relevant biological periodic strains to individual cells in a single experiment.