Temperature influence on the compression and breakage behaviour of yeast cells.

Industrial biotechnology uses microbial cells to produce a wide range of products. While the genetic and molecular properties of these organisms are well understood, less is known about their mechanical properties. Previous work has established a test procedure for single yeast cells using a nanoindentation instrument equipped with a flat-punch probe, which allows single cells (Saccharomyces cerevisiae) to be compressed between two parallel surfaces. The resulting force-displacement curves clearly showed the bursting of the cells and were used to determine characteristics such as burst force and burst energy. Other studies have investigated the influence of growth conditions and measurement conditions on the mechanical characteristics. The recent study examined the mechanical characteristics according to the temperature during compression. Temperature from 0°C to 25°C has no significant effect on the micromechanical properties. Increasing the temperature up to 35°C causes a reduction in the strength of the cells. At even higher temperatures, up to 50°C, the burst force and burst energy increase significantly. A deformation geometry model was used to calculate the cell wall tensile strength as a function of temperature. The results of these studies may facilitate the identification of efficient conditions for cell disruption and product recovery in downstream biotechnological processes.

4D Force Detection of Cell Adhesion and Contractility.

Mechanical signals establish two-way communication between mammalian cells and their environment. Cells contacting a surface exert forces via contractility and transmit them at the areas of focal adhesions. External stimuli, such as compressive and pulling forces, typically affect the adhesion-free cell surface. Here, we demonstrate the collaborative employment of Fluidic Force Microscopy and confocal Traction Force Microscopy supported by the Cellogram solver to enable a powerful integrated force probing approach, where controlled vertical forces are applied to the free surface of individual cells, while the concomitant deformations are used to map their transmission to the substrate. Force transmission across human cells is measured with unprecedented temporal and spatial resolution, enabling the investigation of the cellular mechanisms involved in the adaptation, or maladaptation, to external mechanical stimuli. Altogether, the system enables facile and precise force interrogation of individual cells, with the capacity to perform population-based analysis.

Nuclear Deformation Causes DNA Damage by Increasing Replication Stress.

Cancer metastasis, i.e., the spreading of tumor cells from the primary tumor to distant organs, is responsible for the vast majority of cancer deaths. In the process, cancer cells migrate through narrow interstitial spaces substantially smaller in cross-section than the cell. During such confined migration, cancer cells experience extensive nuclear deformation, nuclear envelope rupture, and DNA damage. The molecular mechanisms responsible for the confined migration-induced DNA damage remain incompletely understood. Although in some cell lines, DNA damage is closely associated with nuclear envelope rupture, we show that, in others, mechanical deformation of the nucleus is sufficient to cause DNA damage, even in the absence of nuclear envelope rupture. This deformation-induced DNA damage, unlike nuclear-envelope-rupture-induced DNA damage, occurs primarily in S/G2 phase of the cell cycle and is associated with replication forks. Nuclear deformation, resulting from either confined migration or external cell compression, increases replication stress, possibly by increasing replication fork stalling, providing a molecular mechanism for the deformation-induced DNA damage. Thus, we have uncovered a new mechanism for mechanically induced DNA damage, linking mechanical deformation of the nucleus to DNA replication stress. This mechanically induced DNA damage could not only increase genomic instability in metastasizing cancer cells but could also cause DNA damage in non-migrating cells and tissues that experience mechanical compression during development, thereby contributing to tumorigenesis and DNA damage response activation.

Simultaneous measurement of turgor pressure and cell wall elasticity in growing pollen tubes.

Plant growth and morphogenesis are tightly controlled processes of division and expansion of individual cells. To fully describe the factors that influence cell expansion, it is necessary to quantify the counteracting forces of turgor pressure and cell wall stiffness, which together determine whether and how a cell expands. Several methods have been developed to measure these parameters, but most of them provide only values for one or the other, and thus require complex models to derive the missing quantity. Furthermore, available methods for turgor measurement are either accurate but invasive, like the pressure probe; or they lack accuracy, such as incipient plasmolysis or indentation-based methods that rely on information about the mechanical properties of the cell wall. Here, we describe a system that overcomes many of the above-mentioned disadvantages using growing pollen tubes of Lilium longiflorum as a model. By combining non-invasive microindentation and cell compression experiments, we separately measure turgor pressure and cell wall elasticity on the same pollen tube in parallel. Due to the modularity of the setup and the large range of the micro-positioning system, our method is not limited to pollen tubes but could be used to investigate the biomechanical properties of many other cell types or tissues.

Inertial flow focusing: a case study in optimizing cellular trajectory through a microfluidic MEMS device for timing-critical applications.

Although microfluidic micro-electromechanical systems (MEMS) are well suited to investigate the effects of mechanical force on large populations of cells, their high-throughput capabilities cannot be fully leveraged without optimizing the experimental conditions of the fluid and particles flowing through them. Parameters such as flow velocity and particle size are known to affect the trajectories of particles in microfluidic systems and have been studied extensively, but the effects of temperature and buffer viscosity are not as well understood. In this paper, we explored the effects of these parameters on the timing of our own cell-impact device, the µHammer, by first tracking the velocity of polystyrene beads through the device and then visualizing the impact of these beads. Through these assays, we find that the timing of our device is sensitive to changes in the ratio of inertial forces to viscous forces that particles experience while traveling through the device. This sensitivity provides a set of parameters that can serve as a robust framework for optimizing device performance under various experimental conditions, without requiring extensive geometric redesigns. Using these tools, we were able to achieve an effective throughput over 360 beads/s with our device, demonstrating the potential of this framework to improve the consistency of microfluidic systems that rely on precise particle trajectories and timing.

3D-printable cell crowding device enables imaging of live cells in compression.

We designed and fabricated, using low-cost 3D printing technologies, a device that enables direct control of cell density in epithelial monolayers. The device operates by varying the tension of a silicone substrate upon which the cells are adhered. Multiple devices can be manufactured easily and placed in any standard incubator. This allows long-term culturing of cells on pretensioned substrates until the user decreases the tension, thereby inducing compressive forces in plane and subsequent instantaneous cell crowding. Moreover, the low-profile device is completely portable and can be mounted directly onto an inverted optical microscope. This enables visualization of the morphology and dynamics of living cells in stretched or compressed conditions using a wide range of high-resolution microscopy techniques.

Cell Mechanical and Physiological Behavior in the Regime of Rapid Mechanical Compressions that Lead to Cell Volume Change.

Cells respond to mechanical forces by deforming in accordance with viscoelastic solid behavior. Studies of microscale cell deformation observed by high speed video microscopy have elucidated a new cell behavior in which sufficiently rapid mechanical compression of cells can lead to transient cell volume loss and then recovery. This work has discovered that the resulting volume exchange between the cell interior and the surrounding fluid can be utilized for efficient, convective delivery of large macromolecules (2000 kDa) to the cell interior. However, many fundamental questions remain about this cell behavior, including the range of deformation time scales that result in cell volume loss and the physiological effects experienced by the cell. In this study, a relationship is established between cell viscoelastic properties and the inertial forces imposed on the cell that serves as a predictor of cell volume loss across human cell types. It is determined that cells maintain nuclear envelope integrity and demonstrate low protein loss after the volume exchange process. These results define a highly controlled cell volume exchange mechanism for intracellular delivery of large macromolecules that maintains cell viability and function for invaluable downstream research and clinical applications.

Deducing density and strength of nanocrystalline Ta and diamond under extreme conditions from X-ray diffraction.

In situ X-ray diffraction with advanced X-ray sources offers unique opportunities for investigating materials properties under extreme conditions such as shock-wave loading. Here, Singh's theory for deducing high-pressure density and strength from two-dimensional (2D) diffraction patterns is rigorously examined with large-scale molecular dynamics simulations of isothermal compression and shock-wave compression. Two representative solids are explored: nanocrystalline Ta and diamond. Analysis of simulated 2D X-ray diffraction patterns is compared against direct molecular dynamics simulation results. Singh's method is highly accurate for density measurement (within 1%) and reasonable for strength measurement (within 10%), and can be used for such measurements on nanocrystalline and polycrystalline solids under extreme conditions (e.g. in the megabar regime).

Controlled single-cell cyclic compression and transcription analysis: A pilot study.

An innovative platform for the study of the molecular mechanisms at the basis of mechanotransduction has been implemented, developing an experimental approach capable of providing controlled dynamic compression stimuli and retrieving the biomolecular response with single-cell sensitivity. The system provides the ability to perform compression-release cycles on single cells with controlled forces in the nN range and a user-defined repetition rate. Experimental procedures to perform qPCR from a small set of single cells were finely tuned. The experimental platform was tested in the context of bone (cell line hFOB 1.19), a physiological environment highly subjected to mechanical stimuli. Target genes were identified in the literature, based on their involvement in the osteogenesis process or in the bone response to mechanical stimuli. qPCR analysis shows an increase in expression of the chosen targets, and confirms the effectiveness of the presented approach for studying living single cells response to dynamic compression.

Controlling seepage in discrete particle simulations of biological systems.

It is now commonplace to represent materials in a simulation using assemblies of discrete particles. Sometimes, one wishes to maintain the integrity of boundaries between particle types, for example, when modelling multiple tissue layers. However, as the particle assembly evolves during a simulation, particles may pass across interfaces. This behaviour is referred to as 'seepage'. The aims of this study were (i) to examine the conditions for seepage through a confining particle membrane and (ii) to define some simple rules that can be employed to control seepage. Based on the force-deformation response of spheres with various sizes and stiffness, we develop analytic expressions for the force required to move a 'probe particle' between confining 'membrane particles'. We analyse the influence that particle's size and stiffness have on the maximum force that can act on the probe particle before the onset of seepage. The theoretical results are applied in the simulation of a biological cell under unconfined compression.

Mechanical characterization of yeast cells: effects of growth conditions.

UNLABELLED: Industrial biotechnology uses microbiological cells to produce a wide range of products. While the organisms in question are well understood regarding their genetic and molecular properties, less is known about their mechanical properties. Previous work has established a testing procedure for single Saccharomyces cerevisiae cells using a Nanoindenter equipped with a Flat Punch probe, allowing the compression between two parallel surfaces. The resulting force-displacement curves clearly showed the bursting of the cells and served to determine characteristic values such as the bursting force, bursting energy and relative deformation. This study examined the mechanical characteristics of yeast cells under the influence of varying cultivation parameters, namely the pH value, temperature, aeration rate, stirrer speed and culture medium composition. It was observed that only temperature and medium composition showed significant effect on the mechanical properties of the cells. Higher temperatures during cultivation caused lower bursting forces and energies. Further analysis of the data showed that the mechanical characteristics of the cells were only influenced by parameters which also had an influence on the growth rate. In conclusion, higher growth rates result in a lower mechanical strength of the yeast cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on the influence of growth conditions on the mechanical properties of yeast cells. Single cell compression tests on Saccharomyces cerevisiae cells indicate that higher growth rates result in a lower mechanical strength of the cells. As in biotechnological processes mechanical degradation is often part of the downstream process to release the product from the micro-organisms, the knowledge about the mechanical properties of the cells is relevant for process optimization.