Mechanical properties of hybridoma cells in batch culture

Summary Direct measurements throughout a batch culture of bursting force, bursting membrane tension, elastic area compressibility modulus, and the size of single hybridoma cells have been made by a novel micromanipulation technique. It has been found that the bursting membrane tension and compressibility modulus rise significantly in the rapid growth phase, and fall in the death phase. An approach is suggested for relating these mechanical properties to the shear sensitivity of the cells when they are exposed to shear stresses in flow fields. It is shown that reports of changes in hybridoma fragility during batch cultures, as measured using viscometers, might be explained using more fundamental micromanipulation measurements.     

Measurements of elastic moduli of silicone gel substrates with a microfluidic device

Thin layers of gels with mechanical properties mimicking animal tissues are widely used to study the rigidity sensing of adherent animal cells and to measure forces applied by cells to their substrate with traction force microscopy. The gels are usually based on polyacrylamide and their elastic modulus is measured with an atomic force microscope (AFM). Here we present a simple microfluidic device that generates high shear stresses in a laminar flow above a gel-coated substrate and apply the device to gels with elastic moduli in a range from 0.4 to 300 kPa that are all prepared by mixing two components of a transparent commercial silicone Sylgard 184. The elastic modulus is measured by tracking beads on the gel surface under a wide-field fluorescence microscope without any other specialized equipment. The measurements have small and simple to estimate errors and their results are confirmed by conventional tensile tests. A master curve is obtained relating the mixing ratios of the two components of Sylgard 184 with the resulting elastic moduli of the gels. The rigidity of the silicone gels is less susceptible to effects from drying, swelling, and aging than polyacrylamide gels and can be easily coated with fluorescent tracer particles and with molecules promoting cellular adhesion. This work can lead to broader use of silicone gels in the cell biology laboratory and to improved repeatability and accuracy of cell traction force microscopy and rigidity sensing experiments.     

Prediction of mechanical damage to animal cells in turbulence

In previous work a model was proposed for estimation of disruption of animal cells in turbulent capillary flows using information about the hydrodynamics, and cell mechanical properties determined by micromanipulation. The model assumed that the capillary flow consists of a laminar sublayer and a homogeneous turbulent region, and within the latter eddies of sizes similar to or smaller than the cells interact with those cells, causing local surface deformations. The proposed mechanism of cell damage was that such deformations result in an increase in membrane tension and surface energy, and that a cell disrupts when its bursting membrane tension and bursting surface energy are exceeded. The surface energy of the cells was estimated from the kinetic energy of appropriate sized eddies. To test the model, cells were disrupted in turbulent flows in capillaries at mean energy dissipation rates ranging from 800 to 2×10⁴ Wkg^–1. The model assumed that the specific lysis rate is almost independent of the number of passes, which was verified by the experimental data. The implication was that despite the damage the cell mechanical properties did not change markedly during multiple recirculations through the capillaries. On average the model underestimated the cell disruption by about 15%. Although the model gave reasonably good predictions, it lacks proper explanation of the independence of the specific lysis rate on the number of passes. In this paper it is shown that this problem can be resolved in principle by consideration of the localisation of the energy dissipation in turbulent capillary flows. The necessity of further modelling of cell-turbulence interactions is demonstrated.     

Effects of pulsatile flow on cultured vascular endothelial cell morphology

Endothelial cells (EC) appear to adapt their morphology and function to the in vivo hemodynamic environment in which they reside. In vitro experiments indicate that similar alterations occur for cultured EC exposed to a laminar steady-state flow-induced shear stress. However, in vivo EC are exposed to a pulsatile flow environment; thus, in this investigation, the influence of pulsatile flow on cell shape and orientation and on actin microfilament localization in confluent bovine aortic endothelial cell (BAEC) monolayers was studied using a 1-Hz nonreversing sinusoidal shear stress of 40 +/- 20 dynes/cm2 (type I), 1-Hz reversing sinusoidal shear stresses of 20 +/- 40 and 10 +/- 15 dynes/cm2 (type II), and 1-Hz oscillatory shear stresses of 0 +/- 20 and 0 +/- 40 dynes/cm2 (type III). The results show that in a type I nonreversing flow, cell shape changed less rapidly, but cells took on a more elongated shape than their steady flow controls long-term. For low-amplitude type II reversing flow, BAECs changed less rapidly in shape and were always less elongated than their steady controls; however, for high amplitude reversal, BAECs did not stay attached for more than 24 hours. For type III oscillatory flows, BAEC cell shape remained polygonal as in static culture and did not exhibit actin stress fibers, such as occurred in all other flows. These results demonstrate that EC can discriminate between different types of pulsatile flow environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

剪切应力对毛细血管内皮细胞代谢的影响

建立的平行平板流协腔装置适用于研究血管内皮细胞代谢对剪切流场的响应。将培养的人胚肾小球血管单层内皮细胞置于剪应力分别为５＊１０＾－５Ｎ／ｃｍ＾２,１＊１０＾－４Ｎ／ｃｍ＾２和１．５＊１０＾－４Ｎ／ｃｍ＾２的定常层流中剪切２５小时。     

Microfluidic accumulation assay probes attachment of biofilm forming diatom cells

Testing of fouling release (FR) technologies is of great relevance for discovery of the next generation of protective marine coatings. In this paper, an accumulation assay to test diatom interaction under laminar flow with the model organism Navicula perminuta is introduced. Using time lapse microscopy with large area sampling allows determination of the accumulation kinetics of the diatom on three model surfaces with different surface properties at different wall shear stresses. The hydrodynamic conditions within the flow cell are described and a suitable shear stress range to perform accumulation experiments is identified at which statistically significant discrimination of surfaces is possible. The observed trends compare well to published adhesion preferences of N. perminuta. Also, previously determined trends of critical wall shear stresses required for cell removal from the same set of functionalized interfaces shows consistent trends. Initial attachment mediated by extracellular polymeric substances (EPS) present outside the diatoms leads to the conclusion that the FR potential of the tested coating candidates can be deducted from dynamic accumulation experiments under well-defined hydrodynamic conditions. As well as testing new coating candidates for their FR properties, monitoring of the adhesion process under flow provides additional information on the mechanism and geometry of attachment and the population kinetics.     

Wall shear stress distribution along the cusp of a tri-leaflet prosthetic valve

High levels of wall shear stress on the surface of valvular cusps can cause mechanical damage to the blood cells and the cusp surfaces. The shear stresses are also responsible for mechanical failure of prosthetic heart valves. Quatitative measurements of wall shear stress in the vicinity of the leaflets are thus essential for diagnosis of suspected complications and provide important information for the design and fabrication of bioprosthetic heart valves. For this purpose we measured the velocity distribution along the inside wall of the cusps of a tri-leaflet heart valve with a two colour laser Doppler anemometer system. The wall shear stresses on the cusp surface were computed and found to range from 80 to 120 N/m² during the ejection phase. Wall shear stresses of up to 180 N/m² were measured in loci of cusp flexure and the accelerated boundary layer. The results of this study show a correlation between the high shear stress loci and the clinically (animal) observed regions of cusp calcification.     

Modeling of orthokinetic flocculation of Saccharomyces cerevisiae.

This study examined the flocculation behavior of two Saccharomyces cerevisiae strains expressing either Flo1 (LCC1209) genotype or NewFlo (LCC125) phenotype in a laminar flow field by measurement of the fundamental flocculation parameter, the orthokinetic capture coefficient. This orthokinetic capture coefficient was measured as a function of shear rate (5.95-223 s(-1)) and temperature (5-45 degrees C). The capture coefficients of these suspensions were directly proportional to the inverse of shear rate, and exhibited an increase as the temperature was increased to 45 degrees C. The capture coefficient of pronase-treated cells was also measured over similar shear rate and temperature range. A theory, which predicts capture coefficient values due to zymolectin interactions, was simplified from that developed by Long et al. [Biophys. J. 76: (1999) 1112]. This new modified theory uses estimates of: (1) cell wall densities of zymolectins and carbohydrate ligands; (2) cell wall collision contact area; and (3) the forward rate coefficient of binding to predict theoretical capture coefficients. A second model that involves both zymolectin interactions and DLVO forces was used to describe the phenomenon of yeast flocculation at intermediate shear ranges, to explain yeast flocculation in laminar flow.     

Direct measurement of the area expansion and shear moduli of the human red blood cell membrane skeleton.

The area expansion and the shear moduli of the free spectrin skeleton, freshly extracted from the membrane of a human red blood cell (RBC), are measured by using optical tweezers micromanipulation. An RBC is trapped by three silica beads bound to its membrane. After extraction, the skeleton is deformed by applying calibrated forces to the beads. The area expansion modulus K(C) and shear modulus mu(C) of the two-dimensional spectrin network are inferred from the deformations measured as functions of the applied stress. In low hypotonic buffer (25 mOsm/kg), one finds K(C) = 4.8 +/- 2.7 microN/m, mu(C) = 2.4 +/- 0.7 microN/m, and K(C)/mu(C) = 1.9 +/- 1.0. In isotonic buffer, one measures higher values for K(C), mu(C), and K(C)/mu(C), partly because the skeleton collapses in a high-ionic-strength environment. Some data concerning the time evolution of the mechanical properties of the skeleton after extraction and the influence of ATP are also reported. In the Discussion, it is shown that the measured values are consistent with estimates deduced from experiments carried out on the intact membrane and agree with theoretical and numerical predictions concerning two-dimensional networks of entropic springs.     

Effects of extracellular fiber architecture on cell membrane shear stress in a 3D fibrous matrix

Interstitial fluid flow has been shown to affect the organization and behavior of cells in 3D environments in vivo and in vitro, yet the forces driving such responses are not clear. Due to the complex architecture of the extracellular matrix (ECM) and the difficulty of measuring fluid flow near cells embedded in it, the levels of shear stress experienced by cells in this environment are typically estimated using bulk-averaged matrix parameters such as hydraulic permeability. While this is useful for estimating average stresses, it cannot yield insight into how local matrix fiber architecture-which is cell-controlled in the immediate pericellular environment-affects the local stresses imposed on the cell surface. To address this, we used computational fluid dynamics to study flow through an idealized mesh constructed of a cubic lattice of fibers simulating a typical in vitro collagen gel. We found that, in such high porosity matrices, the fibers strongly affect the flow fields near the cell, with peak shear stresses up to five times higher than those predicted by the Brinkman equation. We also found that minor remodeling of the fibers near the cell surface had major effects on the shear stress profile on the cell. These findings demonstrate the importance of fiber architecture to the fluid forces on a cell embedded in a 3D matrix, and also show how small modifications in the local ECM can lead to large changes in the mechanical environment of the cell.     

Estimation of disruption of animal cells by turbulent capillary flow

Disruption of animal cells in turbulent capillary flows has been predicted from a model of cell-hydrodynamic interactions using cell mechanical properties determined by micromanipulation. Eddies of sizes similar to or smaller than the cells are presumed to interact with those cells, causing local surface deformations. The proposed mechanism of cell damage is that such deformations result in an increase in membrane tension and surface energy and that a cell disrupts when its bursting membrane tension and bursting surface energy are exceeded. The surface energy of the cells is estimated from the kinetic energy of appropriately sized eddies. To test the model, cells were disrupted in turbulent flows in capillaries at mean energy dissipation rates up to 2 x 10(4) m(2)/s(3). In all cases the model underestimated the cell disruption by about 15%. Such good agreement implies that the approach of the model to the complicated phenomena of cell turbulence interactions is reasonable. (c) 1993 John Wiley & Sons, Inc.     

Effect of Pluronic F-68 on the mechanical properties of mammalian cells

The mechanical properties of TB/C3 hybridoma cells taken from a continuous culture were measured by micromanipulation. The culture conditions were constant except for the presence or absence of Pluronic F-68 in the medium. It was found that the mean bursting membrane tension and the mean elastic area compressibility modulus of the cells were significantly greater (60% and 120%, respectively) in a medium with 0.05% (w/u) Pluronic F-68 compared to that without Pluronic. Pluronic F-68 therefore affected the strength of the membranes when the cells were exposed to it for a long period of time, i.e., in culture. The short-term effect of Pluronic F-68 on cell strength was also tested by its addition at various levels up to 0.2% (w/v) immediately before the mechanical property measurements. The resulting cell strength depended on the Pluronic concentration, but a significant short-term effect could only be detected above a threshold of 0.1% (w/v). Previous reports on the effect of Pluronic F-68 on animal cell culture are evaluated in the light of these observations.     

Shear stress induced stimulation of mammalian cell metabolism

A flow apparatus has been developed for the study of the metabolic response of anchorage-dependent cells to a wide range of steady and pulsatile shear stresses under well-controlled conditions. Human umbilical vein endothelial cell monolayers were subjected to steady shear stresses of up to 24 dynes/cm(2), and the production of prostacyclin was determined. The onset of flow led to a burst in prostacyclin production which decayed to a long term steady state rate (SSR). The SSR of cells exposed to flow was greater than the basal release level, and increased linearly with increasing shear stress. This study demonstrates that shear stress in certain ranges may not be detrimental to mammalian cell metabolism. In fact, throughout the range of shear stresses studied, metabolite production is maximized by maximizing shear stress.     

A three-dimensional viscoelastic model for cell deformation with experimental verification

A three-dimensional viscoelastic finite element model is developed for cell micromanipulation by magnetocytometry. The model provides a robust tool for analysis of detailed strain/stress fields induced in the cell monolayer produced by forcing one microbead attached atop a single cell or cell monolayer on a basal substrate. Both the membrane/cortex and the cytoskeleton are modeled as Maxwell viscoelastic materials, but the structural effect of the membrane/cortex was found to be negligible on the timescales corresponding to magnetocytometry. Numerical predictions are validated against experiments performed on NIH 3T3 fibroblasts and previous experimental work. The system proved to be linear with respect to cytoskeleton mechanical properties and bead forcing. Stress and strain patterns were highly localized, suggesting that the effects of magnetocytometry are confined to a region extending <10 microm from the bead. Modulation of cell height has little effect on the results, provided the monolayer is >5 micro m thick. NIH 3T3 fibroblasts exhibited a viscoelastic timescale of approximately 1 s and a shear modulus of approximately 1000 Pa.     

Experimental technique of measuring dynamic fluid shear stress on the aortic surface of the aortic valve leaflet

Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. The exact cause and mechanism of the progression of AV calcification is unknown, although mechanical forces have been known to play a role. It is thus important to characterize the mechanical environment of the AV. In the current study, we establish a methodology of measuring shear stresses experienced by the aortic surface of the AV leaflets using an in vitro valve model and adapting the laser Doppler velocimetry (LDV) technique. The valve model was constructed from a fresh porcine aortic valve, which was trimmed and sutured onto a plastic stented ring, and inserted into an idealized three-lobed sinus acrylic chamber. Valve leaflet location was measured by obtaining the location of highest back-scattered LDV laser light intensity. The technique of performing LDV measurements near to biological surfaces as well as the leaflet locating technique was first validated in two phantom flow systems: (1) steady flow within a straight tube with AV leaflet adhered to the wall, and (2) steady flow within the actual valve model. Dynamic shear stresses were then obtained by applying the techniques on the valve model in a physiologic pulsatile flow loop. Results show that aortic surface shear stresses are low during early systole (<5 dyn/cm2) but elevated to its peak during mid to late systole at about 18-20 dyn/cm2. Low magnitude shear stress (<5 dyn/cm2) was observed during early diastole and dissipated to zero over the diastolic duration. Systolic shear stress was observed to elevate only with the formation of sinus vortex flow. The presented technique can also be used on other in vitro valve models such as congenitally geometrically malformed valves, or to investigate effects of hemodynamics on valve shear stress. Shear stress data can be used for further experiments investigating effects of fluid shear stress on valve biology, for conditioning tissue engineered AV, and to validate numerical simulations.     

Heterogeneity Profoundly Alters Emergent Stress Fields in Constrained Multicellular Systems

Stress fields emerging from the transfer of forces between cells within multicellular systems are increasingly being recognized as major determinants of cell fate. Current analytical and numerical models used for the calculation of stresses within cell monolayers assume homogeneous contractile and mechanical cellular properties; however, cell behavior varies by region within constrained tissues. Here, we show the impact of heterogeneous cell properties on resulting stress fields that guide cell phenotype and apoptosis. Using circular micropatterns, we measured biophysical metrics associated with cell mechanical stresses. We then computed cell-layer stress distributions using finite element contraction models and monolayer stress microscopy. In agreement with previous studies, cell spread area, alignment, and traction forces increase, whereas apoptotic activity decreases, from the center of cell layers to the edge. The distribution of these metrics clearly indicates low cell stress in central regions and high cell stress at the periphery of the patterns. However, the opposite trend is predicted by computational models when homogeneous contractile and mechanical properties are assumed. In our model, utilizing heterogeneous cell-layer contractility and elastic moduli values based on experimentally measured biophysical parameters, we calculate low cell stress in central areas and high anisotropic stresses in peripheral regions, consistent with the biometrics. These results clearly demonstrate that common assumptions of uniformity in cell contractility and stiffness break down in postconfluence confined multicellular systems. This work highlights the importance of incorporating regional variations in cell mechanical properties when estimating emergent stress fields from collective cell behavior.     

Plane penetration of uterine muscle by intrauterine shields

A finite element approach is used to predict stress and deformation states of uterine muscle tissue under plane strain indentation by a flexible shield. Realistic, one-dimensional "punch" elements at the shield edges assure that muscle shear stresses remain bounded within experimentally measured values. For typical tissue, bearing pressure, deformation flow fields and edge slip stresses leading to tissue damage are calculated. Penetration depth to shield width ratios are up to 3.0. A piecewise linear, elastic approximation to the highly variable, nonlinear mechanical behavior of the tissue is used. Results are applied to the prediction of possible tissue damage by a flexible shield intrauterine contraceptive device, in place and in equilibrium with typical multiparous uteri.     

An apparatus to study the response of cultured endothelium to shear stress

An apparatus which has been developed to study the response of cultured endothelial cells to a wide range of shear stress levels is described. Controlled laminar flow through a rectangular tube was used to generate fluid shear stress over a cell-lined coverslip comprising part of one wall of the tube. A finite element method was used to calculate shear stresses corresponding to cell position on the coverslip. Validity of the finite element analysis was demonstrated first by its ability to generate correctly velocity profiles and wall shear stresses for laminar flow in the entrance region between infinitely wide parallel plates (two-dimensional flow). The computer analysis also correctly predicted values for pressure difference between two points in the test region of the apparatus for the range of flow rates used in these experiments. These predictions thus supported the use of such an analysis for three-dimensional flow. This apparatus has been used in a series of experiments to confirm its utility for testing applications. In these studies, endothelial cells were exposed to shear stresses of 60 and 128 dynes/cm2. After 12 hr at 60 dynes/cm2, cells became aligned with their longitudinal axes parallel to the direction of flow. In contrast, cells exposed to 128 dynes/cm2 required 36 hr to achieve a similar reorientation. Interestingly, after 6 hr at 128 dynes/cm2, specimens passed through an intermediate phase in which cells were aligned perpendicular to flow direction. Because of its ease and use and the provided documentation of wall shear stress, this flow chamber should prove to be a valuable tool in endothelial research related to atherosclerosis.     

KLF2-dependent, shear stress-induced expression of CD59: a novel cytoprotective mechanism against complement-mediated injury in the vasculature

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm(2)) or disturbed flow (+/- 5 dynes/cm(2) at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm(2). Following 24-48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress.