Hydrodynamic Forces Applied on Intercellular Bonds, Soluble Molecules, and Cell-Surface Receptors

Cells and biomolecules exposed to blood circulation experience hydrodynamic forces that affect their function. We present a methodology to estimate fluid forces and force loading rates applied on cellular aggregates, cell-surface proteins, and soluble molecules. Low Reynolds-number hydrodynamic theory is employed. Selected results are presented for biological cases involving platelets, neutrophils, tumor cells, GpIb-like cell-surface receptors, and plasma von Willebrand factor (vWF)-like soluble proteins. Calculations reveal the following: 1), upon application of constant linear shear, cell aggregates and biomolecules experience time-varying forces due to their tumbling motion. 2), In comparison to neutrophil homotypic aggregates, the maximum force applied on neutrophil-platelet aggregates is approximately threefold lower. Thus, alterations in cell size may dramatically alter adhesion molecule requirement for efficient cell binding. Whereas peak forces on homotypic cell doublets are tensile, shear forces dominate in heterotypic doublets with radius ratio <0.3. 3), The peak forces on platelet GpIb and von Willebrand factor are of comparable magnitude. However, they are orders-of-magnitude lower than those applied on intercellular bonds. Charts are provided to rapidly evaluate the magnitude of hydrodynamic force and rotation time-period occurring in any given experiment. The calculation scheme may find application in studies of vascular biology and receptor biophysics.     

Influence of hydrodynamic shear stress on microcarrier-attached cell growth: Cell line dependency and surfactant protection

Animal cell injuries due to fluid-mechanical forces generated by hydrodynamic mixing in bioreactors could become more detrimental for microcarrier systems. In this work, the effects of cell protective surfactants such as pluronic F68 (F68) and polyvinyl alcohol (PVA) were investigated on microcarrier-grown cells under various hydrodynamic stress conditions. Experimental results indicated that adding 0.2% F68 or 0.2% PVA in media enhanced Vero Cell growth against fluid-mechanical forces, but not CHO-K1 and BHK-21 cells. Although affecting the specific growth rate of Vero cells, hydrodynamic shear forces only slightly influenced CHO-K1 and BHK-21 cells. The cellular sensitivity against fluid-mechanical forces for these three cell lines had the following order: Vero cells > CHO-K1 cells > BHK-21 cells. It seems that, the hydrodynamic effects on microcarrier-grown cells and the effectiveness of surfactant protection are heavily dependent on the culture cell type.     

The effects of mechanical forces on intestinal physiology and pathology

The epithelial and non-epithelial cells of the intestinal wall experience a myriad of physical forces including strain, shear, and villous motility during normal gut function. Pathologic conditions alter these forces, leading to changes in the biology of these cells. The responses of intestinal epithelial cells to forces vary with both the applied force and the extracellular matrix proteins with which the cells interact, with differing effects on proliferation, differentiation, and motility, and the regulation of these effects involves similar but distinctly different signal transduction mechanisms. Although normal epithelial cells respond to mechanical forces, malignant gastrointestinal epithelial cells also respond to forces, most notably by increased cell adhesion, a critical step in tumor metastasis. This review will focus on the phenomenon of mechanical forces influencing cell biology and the mechanisms by which the gut responds these forces in both the normal as well as pathophysiologic states when forces are altered. Although more is known about epithelial responses to force, information regarding mechanosensitivity of vascular, neural, and endocrine cells within the gut wall will also be discussed, as will, the mechanism by which forces can regulate epithelial tumor cell adhesion.     

Growth and death in overagitated microcarrier cell cultures

The effects of hydrodynamic forces on cell growth are investigated for animal cells growing on microcarriers. A reduction in net growth was observed with high levels of agitation. DNA measurements indicated that the reduction in net growth was due entirely to cell death, from hydrodynamic forces. No inhibition or enhancement of cell replication appeared to occur with high levels of agitation.     

Evaluation of intermolecular forces in a circulating system

Intercellular interactions, which are mediated by a variety of complex intercellular molecules through the processes of formation and dissociation of molecular bonds, play a critical role in regulating cellular functions in biological systems. Various approaches are applied to evaluate intercellular or molecular bonding forces. To quantify the intermolecular interaction forces, flow chamber has become a meaningful technique as it can ultimately mimic the cellular microenvironment in vivo under physiological flow conditions. Hydrodynamic forces are usually used to predict the intercellular forces down to the single molecular level. However, results show that only using hydrodynamic force will overestimate up to 30% of the receptor-ligand strength when the non-specific forces such as Derjaguin-Landau-Verway-Overbeek (DLVO) forces become un-neglected. Due to the nature of high ion concentration in the physiological condition, electrostatic force is largely screened which will cause DLVO force unbalanced. In this study, we propose to take account of the DLVO force, including van der Waals (VDW) force and electrostatic force, to predict the intermolecular forces of a cell doublet and cell-substrate model in a circulating system. Results also show that the DLVO force has a nonlinear effect as the cell-cell or cell-substrate distance changes. In addition, we used the framework of high accuracy hydrodynamic theories proved in colloidal systems. It is concluded that DLVO force could not be ignored in quantitative studies of molecular interaction forces in circulating system. More accurate prediction of intercellular forces needs to take account of both hydrodynamic force and DLVO force.     

Reversible and irreversible adhesion of motile Escherichia coli cells analyzed by total internal reflection aqueous fluorescence microscopy

The initial events in bacterial adhesion are often explained as resulting from electrostatic and van der Waals forces between the cell and the surface, as described by DLVO theory (developed by Derjaguin, Landau, Verwey, and Overbeek). Such a theory predicts that negatively charged bacteria will experience greater attraction toward a negatively charged surface as the ionic strength of the medium is increased. In the present study we observed both smooth-swimming and nonmotile Escherichia coli bacteria close to plain, positively, and hydrophobically coated quartz surfaces in high- and low-ionic-strength media by using total internal reflection aqueous fluorescence microscopy. We found that reversibly adhering cells (cells which continue to swim along the surface for extended periods) are too distant from the surface for this behavior to be explained by DLVO-type forces. However, cells which had become immobilized on the surface did seem to be affected by electrostatic interactions. We propose that the "force" holding swimming cells near the surface is actually the result of a hydrodynamic effect, causing the cells to swim at an angle along the glass, and that DLVO-type forces are responsible only for the observed immobilization of irreversibly adhering cells. We explain our observations within the context of a conceptual model in which bacteria that are interacting with the surface may be thought of as occupying one of three compartments: bulk fluid, near-surface bulk, and near-surface constrained. A cell in these compartments feels either no effect of the surface, only the hydrodynamic effect of the surface, or both the hydrodynamic and the physicochemical effects of the surface, respectively.     

Flow-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds.

Fixed spherical swollen human red blood cells of blood type B adhering on a glass surface through antigen-antibody bonds to monoclonal mouse antihuman IgM, adsorbed or covalently linked on the surface, were detached by known hydrodynamic forces created in an impinging jet. The dynamic process of detachment of the specifically bound cells was recorded and analyzed. The fraction of adherent cells remaining on the surface decreased with increasing hydrodynamic force. For an IgM coverage of 0.26%, a tangential force on the order of 100 pN was able to detach almost all of the cells from the surface within 20 min. After a given time of exposure to hydrodynamic force, the fraction of adherent cells remaining increased with time, reflecting an increase in adhesion strength. The characteristic time for effective aging was approximately 4 h. Results from experiments in which the adsorbed antibody molecules were immobilized through covalent coupling and from evanescent wave light scattering of adherent cells, imply that deformation of red cells at the contact area was the principal cause for aging, rather than local clustering of the antibody through surface diffusion. Experiments with latex beads specifically bound to red blood cells suggest that, instead of breaking the antigen-antibody bonds, antigen molecules were extracted from the cell membrane during detachment.     

Cell detachment model for an antibody-based microfluidic cancer screening system

We consider cells bound to the floor of a microfluidic channel and present a model of their flow-induced detachment. We approximate hydrodynamic force and cell elastic response using static finite-element simulation of a single cell. Detachment is assumed to occur when hydrodynamic and adhesive forces are roughly equal. The result is extended to multiple cells at the device level using a sigmoidal curve fit. The model is applied to a microfluidic cancer-screening device that discriminates between normal epithelial cells and cells infected with human papillomavirus (HPV), on the basis of increased expression of the transmembrane protein alpha6 integrin in the latter. Here, the cells to be tested are bound to a microchannel floor coated with anti alpha6 integrin antibodies. In an appropriate flow rate range, normal cells are washed away while HPV-infected cells remain bound. The model allows interpolation between data points to choose the optimal flow rate and provides insight into interaction of cell mechanical properties and the flow-induced detachment mechanism. Notably, the results suggest a significant influence of cell elastic response on detachment.     

Modelling Biological Gel Contraction by Cells: Mechanocellular Formulation and Cell Traction Force Quantification

Traction forces developed by most cell types play a significant role in the spatial organisation of biological tissues. However, due to the complexity of cell-extracellular matrix interactions, these forces are quantitatively difficult to estimate without explicitly considering cell properties and extracellular mechanical matrix responses. Recent experimental devices elaborated for measuring cell traction on extracellular matrix use cell deposits on a piece of gel placed between one fixed and one moving holder. We formulate here a mathematical model describing the dynamic behaviour of the cell-gel medium in such devices. This model is based on a mechanical force balance quantification of the gel visco-elastic response to the traction forces exerted by the diffusing cells. Thus, we theoretically analyzed and simulated the displacement of the free moving boundary of the system under various conditions for cells and gel concentrations. This modelis then used as the theoretical basis of an experimental device where endothelial cells are seeded on a rectangular biogel of fibrin cast between two floating holders, one fixed and the other linked to a force sensor. From a comparison of displacement of the gel moving boundary simulated by the model and the experimental data recorded from the moving holder displacement, the magnitude of the traction forces exerted by the endothelial cell on the fibrin gel was estimated for different experimental situations. Different analytical expressions for the cell traction term are proposed and the corresponding force quantifications are compared to the traction force measurements reported for various kind of cells with the use of similar or different experimental devices. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biomechanics of leukocyte rolling

Leukocyte rolling on endothelial cells and other P-selectin substrates is mediated by P-selectin binding to P-selectin glycoprotein ligand-1 expressed on the tips of leukocyte microvilli. Leukocyte rolling is a result of rapid, yet balanced formation and dissociation of selectin-ligand bonds in the presence of hydrodynamic shear forces. The hydrodynamic forces acting on the bonds may either increase (catch bonds) or decrease (slip bonds) their lifetimes. The force-dependent 'catch-slip' bond kinetics are explained using the 'two pathway model' for bond dissociation. Both the 'sliding-rebinding' and the 'allosteric' mechanisms attribute 'catch-slip' bond behavior to the force-induced conformational changes in the lectin-EGF domain hinge of selectins. Below a threshold shear stress, selectins cannot mediate rolling. This 'shear-threshold' phenomenon is a consequence of shear-enhanced tethering and catch bond-enhanced rolling. Quantitative dynamic footprinting microscopy has revealed that leukocytes rolling at venular shear stresses (>0.6 Pa) undergo cellular deformation (large footprint) and form long tethers. The hydrodynamic shear force and torque acting on the rolling cell are thought to be synergistically balanced by the forces acting on tethers and stressed microvilli, however, their relative contribution remains to be determined. Thus, improvement beyond the current understanding requires in silico models that can predict both cellular and microvillus deformation and experiments that allow measurement of forces acting on individual microvilli and tethers.     

Two-Bubble Acoustic Tweezing Cytometry for Biomechanical Probing and Stimulation of Cells

The study of mechanotransduction relies on tools that are capable of applying mechanical forces to elicit and assess cellular responses. Here we report a new (to our knowledge) technique, called two-bubble acoustic tweezing cytometry (TB-ATC), for generating spatiotemporally controlled subcellular mechanical forces on live cells by acoustic actuation of paired microbubbles targeted to the cell adhesion receptor integrin. By measuring the ultrasound-induced activities of cell-bound microbubbles and the actin cytoskeleton contractile force responses, we determine that TB-ATC elicits mechanoresponsive cellular changes via cyclic, paired displacements of integrin-bound microbubbles driven by the attractive secondary acoustic radiation force (sARF) between the bubbles in an ultrasound field. We demonstrate the feasibility of dual-mode TB-ATC for both subcellular probing and mechanical stimulation. By exploiting the robust and unique interaction of ultrasound with microbubbles, TB-ATC provides distinct advantages for experimentation and quantification of applied forces and cellular responses for biomechanical probing and stimulation of cells.     

Effect of viscoelasticity on the analysis of single-molecule force spectroscopy on live cells

Single-molecule force spectroscopy is used to probe the kinetics of receptor-ligand bonds by applying mechanical forces to an intermediate media on which the molecules reside. When this intermediate media is a live cell, the viscoelastic properties can affect the calculation of rate constants. We theoretically investigate the effect of media viscoelasticity on the common assumption that the bond force is equal to the instantaneous applied force. Dynamic force spectroscopy is simulated between two cells of varying micromechanical properties adhered by a single bond with a constant kinetic off-rate. We show that cell and microvilli deformation, and hydrodynamic drag contribute to bond forces that can be 28-90% lower than the applied force for loading rates of 10(3)-10(7) pN/s, resulting in longer bond lifetimes. These longer bond lifetimes are not caused by changes in bond kinetics; rather, they are due to the mechanical response of the intermediate media on which the bonds reside. Under the assumption that the instantaneous bond force is equal to the applied force--thereby ignoring viscoelasticity--leads to 14-39% error in the determination of off-rates. We present an approach that incorporates viscoelastic properties in calculating the instantaneous bond force and kinetic dissociation parameter of the intermolecular bond.     

Transient relative motion of two cells in a channel flow

The hydrodynamic interaction of a red blood cell and a white blood cell in microvessels is studied, by use of a two-dimensional numerical model. The red blood cell, modeled as a small rigid circular cylinder, and the white blood cell, modeled as a larger rigid circular cylinder, are immersed in an incompressible Newtonian fluid in a two-dimensional channel. It is assumed that no external force or moment acts on the model cells, and the effect of inertia forces on the motion of the fluid and the cells is neglected. The velocity field of the suspending fluid and the instantaneous velocities of the two model cells are computed by the finite element method. Using the translational velocities of the model cells obtained, the trajectories of their relative motion are determined, for various initial positions. It is shown that the cells may or may not pass each other or separate, depending on the initial positions. The present results compare well to the experimental results.     

Hydrodynamic shear forces increase Japanese encephalitis virus production from microcarrier-grown Vero cells

Understanding the effect of hydrodynamic shear forces on microcarrier-attached cells is critical in several viral vaccine production processes, owing to that only the anchorage-dependent cells can be used for virus propagation in cultures. This study demonstrated that increasing the hydrodynamic shear forces in microcarrier cultures can increase the production of a vaccine strain of Japanese encephalitis virus (on a per cell basis) in Vero cells but not BHK-21. The shear force-enhanced JEV production were highly effective at around 2-3 d post infection and required the concentration of fetal bovine serum supplemented in medium above 2.5%. To our knowledge, this study reports for the first time that increasing the hydrodynamic shear forces on microcarrier-grown cells increases virus production in agitated bioreactor cultures.     

Separation and characterization of red blood cells with different membrane deformability using steric field-flow fractionation

Human red blood cells were treated in different ways to alter their membrane deformability, and the hydrodynamic behavior of these altered cells was studied using the steric field-flow fractionation (FFF) technique. The relationships between cell retention in the FFF channel, flow-rate of the carrier fluid and the applied field strength were studied for normal and glutaraldehyde-fixed human red cells, and separation conditions were optimized. The effect of flow-induced hydrodynamic lift forces on red cell retention in the steric FFF channel was studied, and the results suggest that the membrane deformability of the red cell is an important factor contributing to the lift force, besides other previously described effects due to density and flow velocity. Using steric FFF, a mixture of normal and glutaraldehyde-fixed human red cells was completely separated with a resolution twice that found in published d ata from gel permeation, another hydrodynamic separation technique. Partial loss of membrane deformability, induced by different degrees of glutaraldehyde-fixation, by diamide, or by a thermal treatment, has also been studied. Steric FFF is thus shown to have potential for rapid separation and differentiation of red cells with different density and membrane deformability, conditions known to be associated with, e.g., cell senescence and certain hematological diseases.     

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitrofrequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.     

Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.     

Intercellular mechanotransduction: cellular circuits that coordinate tissue responses to mechanical loading

Physical forces play an important role in modulating cell function and shaping tissue structure. Mechanotransduction, the process by which cells transduce physical force-induced signals into biochemical responses, is critical for mediating adaptations to mechanical loading in connective tissues. While much is known about mechanotransduction in cells involving forces delivered through extracellular matrix proteins and integrins, there is limited understanding of how mechanical signals are propagated through the interconnected cellular networks found in tissues and organs. We propose that intercellular mechanotransduction is a critical component for achieving coordinated remodeling responses to force application in connective tissues. We examine here recent evidence on different pathways of intercellular mechanotransduction and suggest a general model for how multicellular structures respond to mechanical loading as an integrated unit.     

Mechanotransduction in blood cells

Blood cells are subjected to various mechanical forces; including pressure, flow, shear force, gravity, and forces acting against them with varying stiffness (eg. blood vessel wall). Scientists have discovered that these forces have profound effects on cellular growth, differentiation, secretion of cytokines, cell death, and migration. These processes are called mechanotransduction, a conversion of mechanical forces to biochemical signals. In this article the author reviews biophysical forces that affect biological functions of blood cells and their responses in normal physiology and pathophysiology. Although input (forces) and output (cellular responses) have been well studied by utilizing recently developed various force-generating devices, the molecular mechanism of mechanotransudction is still a mystery. This is because reconstructing molecular interaction in the presence of mechanical forces in vitro is highly challenging and until now the molecular dynamics involved in structural changes caused by these forces are largely unknown. Nevertheless, the author has reviewed a few examples of potential structural effects on the molecular mechanism of mechanotransduction.