Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 μm diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young''s modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.     

A computational model for the transit of a cancer cell through a constricted microchannel

We propose a three-dimensional computational model to simulate the transient deformation of suspended cancer cells flowing through a constricted microchannel. We model the cell as a liquid droplet enclosed by a viscoelastic membrane, and its nucleus as a smaller stiffer capsule. The cell deformation and its interaction with the suspending fluid are solved through a well-tested immersed boundary lattice Boltzmann method. To identify a minimal mechanical model that can quantitatively predict the transient cell deformation in a constricted channel, we conduct extensive parametric studies of the effects of the rheology of the cell membrane, cytoplasm and nucleus and compare the results with a recent experiment conducted on human leukaemia cells. We find that excellent agreement with the experiment can be achieved by employing a viscoelastic cell membrane model with the membrane viscosity depending on its mode of deformation (shear versus elongation). The cell nucleus limits the overall deformation of the whole cell, and its effect increases with the nucleus size. The present computational model may be used to guide the design of microfluidic devices to sort cancer cells, or to inversely infer cell mechanical properties from their flow-induced deformation.

     

An elasto-visco-plastic model of cell aggregates

Concentrated cell suspensions exhibit different mechanical behavior depending on the mechanical stress or deformation they undergo. They have a mixed rheological nature: cells behave elastically or viscoelastically, they can adhere to each other whereas the carrying fluid is usually Newtonian. We report here on a new elasto-visco-plastic model which is able to describe the mechanical properties of a concentrated cell suspension or aggregate. It is based on the idea that the rearrangement of adhesion bonds during the deformation of the aggregate is related to the existence of a yield stress in the macroscopic constitutive equation. We compare the predictions of this new model with five experimental tests: steady shear rate, oscillatory shearing tests, stress relaxation, elastic recovery after steady prescribed deformation, and uniaxial compression tests. All of the predictions of the model are shown to agree with these experiments.     

Enhanced antibacterial properties and the cellular response of stainless steel through friction stir processing

Biofilm related bacterial infection is one of the primary causes of implant failure. Limiting bacterial adhesion and colonization of pathogenic bacteria is a challenging task in health care. Here, a highly simplistic processing technique for imparting antibacterial properties on a biomedical grade stainless steel is demonstrated. Low-temperature high strain-rate deformation achieved using submerged friction stir processing resulted in a nearly single phase ultra-fine grain structure. The processed stainless steel demonstrated improved antibacterial properties for both Gram-positive and Gram-negative bacteria, significantly impeding biofilm formation during the in vitro study. Also, the processed stainless steel showed better compatibility with human fibroblasts manifested through apparent cell spreading and proliferation. The substantial antibacterial properties of the processed steel are explained in terms of the favorable electronic characteristics of the metal-oxide and by using classical Derjaguin–Landau–Verwey–Overbeek (DLVO) and the extended DLVO (XDLVO) approach at the cell–substrate interface.     

A two-layer outer hair cell model with orthotropic piezoelectric properties: correlation of cell resonant frequencies with tuning in the cochlea

The frequency response of outer hair cells (OHCs) of different lengths is studied using a mathematical model of a two-layer cylindrical shell with orthotropic properties. Material properties in the model are determined from experimental measurements reported in the literature, and the variation of material properties with the cell length is studied. The cortical lattice's Poisson ratios are found to remain fairly constant with cell length, while its stiffness changes significantly with cell length. The natural frequencies corresponding to several modes of deformation of an OHC with intracellular and extracellular fluids are calculated from this model. Our results suggest that the best frequency in the cochlea at the position where the OHC is located corresponds to different modes of deformation of the OHC, depending on the OHC length. For short OHCs, the best frequency is close to the natural frequency of the axisymmetric mode; for long OHCs, it is close to the natural frequencies of the beam-like bending and pinched modes. Such a difference in resonant modes for short and long OHCs at the best frequency suggests that different modes of OHC elongation motility may be present in amplifying the basilar membrane motion in the high and low frequency regions of the cochlea.     

Effect of degree of uniformity on predicted visual cortical response tuning curves

 The different cortical visual cells exhibit a large repertoire of responses to sinusoidal gratings, depending on their receptive field structure and the stimulation parameters. It has been shown previously that the tuning curves and histogram shapes of cell responses are affected by subunit distances. One receptive field model (Spitzer and Hochstein 1985b) incorporated subunit distance but assigned it as a constant parameter, for ease of calculation. Here we investigate different tuning curve properties of various primary cortical cell types during testing of 10 deg of nonuniform distances of the receptive fields’ subunits. The effect of nonuniformity was compared for average responses, tuning curve shapes, maximum peak responses, and bandwidths across four cell types of different sizes. The shapes and other properties of tuning curves are usually found to be retained also when the degree of uniformity is not very high for most of the cell types. In addition, the effect of uniformity is compared across these different response properties. The maximum peak responses of the tuning curve are found to display a lower coefficient of variation than the bandwidth, for all cell types, for most degrees of uniformity. Received: 15 June 1993/Accepted in revised form: 5 August 1994     

Influence of temperature on the interaction of Concanavalin A with chick fibroblasts during embryo development

The interaction between Concanavalin A and chick embryo fibroblasts was studied. Cells from younger and older embryos had the same number of lectin receptor sites per cell at 4°, 21° and 37°C but the affinity constants increased with increasing temperature. Analysis of the binding data according to Scatchard showed that the apparent changes in binding as a function of temperature might be related to thermodynamic properties. The lectin binding sites on the cell surface proved homogeneous with regard to binding properties.The age-related differences noted in the affinities of the cells to bind Concanavalin A could be related to differences in the degree of rearrangement of the cell surface components and/or to a change in the structure of cell surface glycoconjugates, and may serve to explain the differences in the effect of Concanavalin A on cell growth.     

Mechanical Response of Single Plant Cells to Cell Poking： A Numerical Simulation Model

Cell poking is an experimental technique that is widely used to study the mechanical properties of plant cells. A full understanding of the mechanical responses of plant cells to poking force Is helpful for experimental work. The aim of this study was to numerically investigate the stress distribution of the cell wall, cell turgor, and deformation of plant cells in response to applied poking force. Furthermore, the locations damaged during poking were analyzed. The model simulates cell poking, with the cell treated as a spherical, homogeneous, isotropic elastic membrane, filled with incompressible, highly viscous liquid. Equilibrium equations for the contact region and the non-contact regions were determined by using membrane theory. The boundary conditions and continuity conditions for the solution of the problem were found. The forcedeformation curve, turgor pressure and tension of the cell wall under cell poking conditions were obtained. The tension of the cell wall circumference was larger than that of the meridian. In general, maximal stress occurred at the equator around. When cell deformation increased to a certain level, the tension at the poker tip exceeded that of the equator. Breakage of the cell wall may start from the equator or the poker tip, depending on the deformation. A nonlinear model is suitable for estimating turgor, stress, and stiffness, and numerical simulation is a powerful method for determining plant cell mechanical properties.     

Theoretical and experimental studies on viscoelastic properties of erythrocyte membrane.

The deformation of a portion of erythrocyte during aspirational entry into a micropipette has been analyzed on the basis of a constant area deformation of an infinite plane membrane into a cylindrical tube. Consideration of the equilibrium of the membrane at the tip of the pipette has generated the relation between the aspirated length and the dimensionless time during deformational entry as well as during relaxation after the removal of aspiration pressure. Experimental studies on deformation and relaxation of normal human erythrocytes were performed with the use of micropipettes and a video dimension analyzer which allowed the continuous recording of the time-courses. The deformation consisted of an initial rapid phase with a membrane viscosity (range 0.6 x 10(-4) to 4 x 10(-4) dyn.s/cm) varying inversely with the degree of deformation and a later slow phase with a high membrane viscosity (mean 2.06 x 10(-2) dyn.s/cm) which was not correlated with the degree of deformation. The membrane viscosity of the recovery phase after 20 s of deformation (mean 5.44 x 10(-4) dyn.s/cm) was also independent of the degree of deformation. When determined after a short period of deformation (e.g., 2 s), however, membrane viscosity of the recovery phase became lower and agreed with that of the deformation phase. These results suggest that the rheological properties of the membrane can undergo dynamic changes depending on the extent and duration of deformation, reflecting molecular rearrangement in response to membrane strain.     

Membrane-protective properties of isobornylphenols-a new class of antioxidants

The membrane protective and membrane active properties and the antioxidative activity of new semisynthetic antioxidants—isobornylphenols were studied. The presence of oxidant and cytotoxic properties of the compounds were evaluated considering the degree of hemolysis of erythrocytes, either spontaneous or induced by hydrogen peroxide. All the studied compounds were found to have significant antioxidative activity in certain conditions. But their capacity to protect membrane erythrocytes from oxidative stress substantially depended on the structure and concentration of the compound. The highest membrane protective activity was observed for 2,6-diisobornyl-4-methylphenol, which has isobornyl in both of its ortho-positions. Scanning electron microscopy of blood erythrocyte surface architectonics confirmed the ability of the studied compounds to interact with the cell membrane and to change its structure. A relationship between erythrocyte morphological transformation according to bilayer-couple hypothesis depending on isobornylphenols membrane behavior and the cytotoxic effect of certain compound high concentrations reflected in low membrane protective activity in the model cell system was shown. The data obtained allow us to conclude that the biological activity of isobornylphenols is due to both their antioxidative properties and their ability to interact with the cell membranes.     

Biomechanical consequences of developmental changes in trabecular architecture and mineralization of the pig mandibular condyle

The purpose of the present study was to examine the changes in apparent mechanical properties of trabecular bone in the mandibular condyle during fetal development and to investigate the contributions of altering architecture, and degree and distribution of mineralization to this change. Three-dimensional, high-resolution micro-computed tomography (microCT) reconstructions were utilized to assess the altering architecture and mineralization during development. From the reconstructions, inhomogeneous finite element models were constructed, in which the tissue moduli were scaled to the local degree of mineralization of bone (DMB). In addition, homogeneous models were devised to study the separate influence of architectural and DMB changes on apparent mechanical properties. It was found that the bone structure became stiffer with age. Both the mechanical and structural anisotropies pointed to a rod-like structure that was predominantly oriented from anteroinferior to posterosuperior. Resistance against shear, also increasing with age, was highest in the sagittal plane. The reorganization of trabecular elements, which occurred without a change in bone volume fraction, contributed to the increase in apparent stiffness. The increase in DMB, however, contributed more dominantly. Incorporating the observed inhomogeneous distribution of mineralization decreased the apparent stiffness, but increased the mechanical anisotropy. This denotes that there might be a directional dependency of the DMB of trabecular elements, i.e. differently orientated trabecular elements might have different DMBs. In conclusion, the changes in DMB and its distribution are important to consider when studying mechanical properties during development and should be considered in other situations where differences in DMB are expected.     

Fiber orientation effects in simple shearing of fibrous soft tissues

Fiber-reinforcement is a common feature of many soft biological tissues. Continuum mechanics modeling of the mechanical response of such tissues using transversely isotropic hyperelasticity is now well developed. The fundamental deformation of simple shear within this framework is examined here. It is well known that the normal stress effect characteristic of nonlinear elasticity plays a crucial role in maintaining a homogeneous deformation state in the bulk of the specimen. Here we consider the effect of anisotropy and fiber-orientation on the shear and normal stresses. It is shown that the confining traction that needs to be applied to the top and bottom faces of a block in order to maintain simple shear can be compressive or tensile depending on the degree of anisotropy and on the angle of orientation of the fibers. In the absence of such an applied traction, an unconfined sample tends to bulge outwards or contract inwards perpendicular to the direction of shear so that one has the possibility of both a positive or negative Poynting effect. The results are illustrated using experimental data obtained by other authors for porcine brain white matter. The general results obtained here are relevant to the development of accurate shear test protocols for the determination of constitutive properties of fibrous biological soft tissues.     

Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension.

We have investigated how extracellular matrix (ECM) alters the mechanical properties of the cytoskeleton (CSK). Mechanical stresses were applied to integrin receptors on the apical surfaces of adherent endothelial cells using RGD-coated ferromagnetic microbeads (5.5-microns diameter) in conjunction with a magnetic twisting device. Increasing the number of basal cell-ECM contacts by raising the fibronectin (FN) coating density from 10 to 500 ng/cm2 promoted cell spreading by fivefold and increased CSK stiffness, apparent viscosity, and permanent deformation all by more than twofold, as measured in response to maximal stress (40 dyne/cm2). When the applied stress was increased from 7 to 40 dyne/cm2, the stiffness and apparent viscosity of the CSK increased in parallel, although cell shape, ECM contacts, nor permanent deformation was altered. Application of the same stresses over a lower number ECM contacts using smaller beads (1.4-microns diameter) resulted in decreased CSK stiffness and apparent viscosity, confirming that this technique probes into the depth of the CSK and not just the cortical membrane. When magnetic measurements were carried out using cells whose membranes were disrupted and ATP stores depleted using saponin, CSK stiffness and apparent viscosity were found to rise by approximately 20%, whereas permanent deformation decreased by more than half. Addition of ATP (250 microM) under conditions that promote CSK tension generation in membrane-permeabilized cells resulted in decreases in CSK stiffness and apparent viscosity that could be detected within 2 min after ATP addition, before any measurable change in cell size.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ektacytometry: a method for characterizing erythrocyte deformability

In principle, the ektacytometer consists of a combination of a Laser-illuminated diffractometer with a circular viscometric fluid flow, analogous to that of a rotation viscometer. In this device erythrocytes are deformed to ellipsoid-like shapes and produce a diffraction pattern which, depending on the flow conditions, is to some degree elliptically distorted. From the shape of this pattern the extent of cell deformation can be deduced. In this survey an introduction into the mode of operation of the ektacytometer as well as an overview on methodical variants are given on the basis of literature. Since its publication in 1974 ektacytometry has found many applications, mainly in basic research. Presumably, it will receive still broader interest also as a routine method in haematological or pharmaceutical laboratories.     

The mechanical behaviour of cancellous bone

Cancellous bone has a cellular structure: it is made up of a connected network of rods and plates. Because of this, its mechanical behaviour is similar to that of other cellular materials such as polymeric foams. A recent study on the mechanisms of deformation in such materials has led to an understanding of how their mechanical properties depend on their relative density, cell wall properties and cell geometry. In this paper, the results of this previous study are applied to cancellous bone in an attempt to further understand its mechanical behaviour. The results of the analysis agree reasonably well with experimental data available in the literature.     

Polygalacturonase-inhibiting protein is a structural component of plant cell wall

It is generally believed that plants "evolved a strategy of defending themselves from a phytopathogen attack" during evolution. This metaphor is used frequently, but it does not facilitate understanding of the mechanisms providing plant resistance to the invasion of foreign organisms and to other unfavorable external factors, as well as the role of these mechanisms in plant growth and development. Information on processes involving one of the plant resistance factors--polygalacturonase-inhibiting protein (PGIP)--is considered in this review. The data presented here indicate that PGIP, being an extracellular leucine-rich repeat-containing protein, performs important functions in the structure of plant cell wall. Amino acid residues participating in PGIP binding to homogalacturonan in the cell wall have been determined. The degree of methylation and the mode of distribution of homogalacturonan methyl groups are responsible for the formation of a complex structure, which perhaps determines the specificity of PGIP binding to pectin. PGIP is apparently one of the components of plant cell wall determining some of its mechanical properties; it is involved in biochemical processes related to growth, expansion, and maceration, and it influences plant morphology. Polygalacturonase (PG) is present within practically all plant tissues, but the manifestation of its activity varies significantly depending on physiological conditions in the tissue. Apparently, the regulation of PG functioning in apoplast significantly affects the development of processes associated with the modification of the structure of plant cell wall. PGIP can regulate PG activity through binding to homogalacturonan. The genetically determined structure of PGIP in plants determines the mode of its interaction with an invader and perhaps is one of the factors responsible for the set of pathogens causing diseases in a given plant species.     

Survival of biological cells deformed in a narrow gap

Recent studies show that during slow freezing of biological cells, the cells may be also injured by not only chemical damage but also mechanical damage induced by ice crystal compression. A new experimental procedure is developed to quantify cell destruction by deformation with two parallel surfaces. The viability of cells (prostatic carcinoma cells, 17.5 microns in mean diameter) is measured as a function of gap size ranging from 3.5 microns to 16.2 microns at 0 degree C, 23 degrees C and 37 degrees C. The viability at a smaller gap size is significantly lower at 37 degrees C than at 23 degrees C, while the difference between 0 degree C and 23 degrees C is much smaller. This suggests that deformation damage is related to the deformation of the cytoskeleton rather than the mechanical properties of the lipid membrane.     

The mechanical behavior of isolated Avena coleoptile walls subjected to constant stress: properties and relation to cell elongation

In order to assess the role of the mechanical properties of the wall in auxin-induced cell elongation, a study has been made of the ability of isolated Avena coleoptile walls to extend (creep) when subjected to a constant applied stress. Creep occurs as a viscoelastic extension which has the following characteristics: the extension is proportional to log time and is partly reversible, and the extension rate has a Q₁₀ of about 1.05 and is markedly greater in auxin-pretreated walls. In nonconditioned walls the extension rate is proportional to applied stress, but pre-extension causes the appearance of an apparent yield strain. The similarity of creep and instantaneous plastic deformation in response to temperature or to pretreatment with auxin or KCN suggests that the instantaneous deformation is simply the viscoelastic extension which occurs at very short times. A comparison of these viscoelastic properties with the properties of auxin-induced cell elongation indicates that cell elongation requires more than just a physical extension of the wall. It is suggested that elongation occurs as a series of extension steps, each of which involves a viscoelastic extension preceded or accompanied by an auxin-dependent biochemical change in the wall properties.     

Operando Visualization of Morphological Dynamics in All‐Solid‐State Batteries

All‐solid‐state batteries (SSBs) are considered as attractive options for next‐generation energy storage owing to the favorable properties (unit transference number and thermal stabilities) of solid electrolytes. However, there are also serious concerns about mechanical deformation of solid electrolytes leading to the degradation of the battery performance. Therefore, understanding the mechanism underlying the electromechanical properties in SSBs is essentially important. Here, 3D and time‐resolved measurements of an all‐solid‐state cell using synchrotron radiation X‐ray tomographic microscopy are shown. The gradient of the electrochemical reaction and the morphological evolution in the composite layer can be clearly observed. Volume expansion/compression of the active material (Sn) is strongly oriented along the thickness of the electrode. While this results in significant deformation (cracking) in the solid electrolyte region, organized cracking patterns depending on the particle size and their arrangements is also found. This study based on operando visualization therefore opens the door toward rational design of particles and electrode morphology for all‐solid‐state batteries.