Influence of glutaraldehyde fixation of cells adherent to solid substrata on their detachment during exposure to shear stress

In order to determine the response of fixed and nonfixed cells adherent to a solid substratum to shear stress, human fibroblasts were allowed to adhere and spread on either hydrophilic glass or hydrophobic Fluoroethylene-propylene (FEP-Teflon) and fixed with glutaraldehyde. Then, the cells were exposed to an incrementally loaded shear stress in a parallel plate flow chamber up to shear stresses of about 500 dynes/cm², followed by exposure to a liquid-air interface passage. The cellular detachment was compared with the one of nonfixed cells. In case of fixed cells, 50% of the adhering cells detached from FEP-Teflon at a shear stress of 350 dynes/cm², whereas 50% of the adhering, nonfixed cells detached already at a shear stress of 20 dynes/cm². No fixed cells detached from glass for shear stresses up to at least 500 dynes/cm². More than 50% of the nonfixed cells were detached from glass at a shear stress of 350 dynes/cm². Furthermore, the shape and morphology of fixed cells did not change during the incrementally loaded flow, in contrast to the ones of nonfixed cells, which clearly rounded up prior to detachment.     

Influence of shear stress on adherent mammalian cells during division

Summary The influence of shear stress (0 to 4.5 N m^–2) on morphology and adherence of anchorage-dependent baby hamster kidney (BHK 21 cl3) cells during cell division was investigated by means of a time lapse film. With increasing shear stress level divided cells needed more time for spreading.     

Influence of the temperature on the shear stress sensitivity of adherent BHK 21 cells

The influence of temperature on the shear sensitivity of anchorage-dependent baby hamster kidney (BHK) cells was investigated. The temperature effect in general was compared for stressed and unstressed cells. Both the growth rate as well as the shear sensitivity are temperature-dependent. Decreasing the temperature lowered the growth rate and increased the ability of the BHK cells to withstand shear stress. Correspondence to: G. Kretzmer     

Direct measurement of shear strain in adherent vascular endothelial cells exposed to fluid shear stress

Functional and morphological responses of endothelial cells (ECs) to fluid shear stress are thought to be mediated by several mechanosensitive molecules. However, how the force due to fluid shear stress applied to the apical surface of ECs is transmitted to the mechanosensors is poorly understood. In the present paper, we performed an analysis of an intracellular mechanical field by observation of the deformation behaviors of living ECs exposed to shear stress with a novel experimental method. Lateral images of human umbilical vein ECs before and after the onset of flow were obtained by confocal microscopy, and image correlation and finite element analysis were performed for quantitative analyses of subcellular strain due to shear stress. The shear strain of the cells changed from 1.06 ± 1.09% (mean ± SD) to 4.67 ± 1.79% as the magnitude of the shear stress increased from 2 to 10 Pa. The nuclei of ECs also exhibited shear deformation, which was similar to that observed in cytoplasm, suggesting that nuclei transmit forces from apical to intracellular components, as well as cytoskeletons. The obtained strain-stress relation resulted in a mean shear modulus of 213 Pa for adherent ECs. These results provide a mechanical perspective on the investigation of flow-sensing mechanisms of ECs.     

Determination of a "critical shear stress level" applied to adherent mammalian cells.

An apparatus for the detailed investigation of the influence of shear stress on adherent BHK cells was developed. Shear forces between 0.0 and 2.5 N m-2 were studied. The influence on cell viability, cell morphology, cell lysis, and cell size was determined. Increasing shear forces as well as increasing exposure duration caused increasing changes in cell morphology and cell death. A "critical shear stress level" was determined.     

Supravital exposure to propidium iodide identifies apoptosis on adherent cells

BACKGROUND: Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.     

The effects of shear stress and metastatic phenotype on the detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal, transformed, and reverted rat fibroblasts from a confluent monolayer of normal fibroblasts. In this method, known shear stresses were applied to the adherent cells and the percent of cells detached from the monolayer was determined. Results indicate that the detachment of all cell types increased with increasing shear stress and detachment of highly metastatic ras-transformed cells was significantly higher than that of either nonmetastatic normal cells or transformed cells reverted with the Kirsten ras revertant (K-rev 1a) gene, which are lowly metastatic. From these results, it is concluded that a correlation exists between the metastatic phenotype of the cell and its ability to detach from normal cells.     

Vitronectin enhances adhesion force and t-PA production of weakly adherent 293 cells exposed to a shear stress

The effects of shear stress on the adhesion andproductivity of 293 cells were studied quantitativelyand compared with those of Vero and human liver cells.These cells were cultured in polystyrene dishes byusing shear stress exposing equipment. 50% of 293cells cultured in 2% FBS supplemented medium detachedfrom the dish after 29 h of exposure to a shear stressof 0.10 Pa. On the other hand, 90% of Vero and humanliver cells remained on the dish under the samecondition. Observations with scanning electronmicroscopy about cell adhesion plaques on the surfaceof the dish showed that the area covered withlamellipodia and the number of microspikes for 293cells were found to be less than those of the othercell lines. Several attachment factors, especiallyvitronectin, were found to enhance the number ofmicrospikes and the adhesion force of 293 cells.Almost 100% of 293 cells remained on thevitronectin-coated dish after 40 h under 0.10 Pa ofshear stress. A higher shear stress (greater than 0.10Pa) caused a decrease in tissue plasminogen activator(t-PA) productivity of 293 cells. But 0.03 Pastimulated the t-PA secretion on the non-coated dish.Vitronectin also enhanced the t-PA secretion evenunder 0.10 Pa. These results indicate that theadhesion force of 293 cells is obviously weaker thanthat of the other cell lines, and vitronectin enhancesthe adhesion force and the productivity of 293 cellsexposed to a shear stress.     

Influence of lactate, ammonia, and osmotic stress on adherent and suspension BHK cells.

Adherent and suspension Baby Hamster Kidney (BHK) 21c13 cells were cultivated in a 2.5-1 stirred-tank reactor with indirect aeration. Cell concentration and viability as well as glucose, lactate, ammonia, and protein concentrations in the medium and intracellular and extracellular activities of the intracellular enzymes were determined off-line. The concentrations of glucose, lactate, ammonia, and the activity of lactate dehydrogenase in the culture medium were monitored on-line. The cell/cell fragment size distribution was determined by laser flow cytometer off-line. In several runs, the size distributions were ascertained on-line by a laser flow cytometer. The influence of lactate, ammonia, and osmotic pressure on the viability and biological parameters of the suspension cells was evaluated. In Roux flasks, lactate and ammonia had considerable influence on the cell properties; in stirred tank reactors, these influences were negligible up to 9.5 g l-1 lactate and 150 mg l-1 NH+4 ion concentrations. The influence of high osmolarity on the biological parameters of the cells was much less in the stirred-tank than in the Roux flasks. The adhesion of adherent cells on a surface was impeded neither by the lactate (up to 6 g l-1) nor by the ammonia concentration (up to 150 mg l-1). However, with increasing osmolarity, the fraction of the cells adhered to a surface reduced to below 5% (at 680 mOsmol l-1).     

Response of mammalian cells to shear stress

Summary The influence of shear forces on adherent mammalian cells was investigated by means of a developed flow chamber. The viability of the cells decreased with increasing exposure level and duration. Additional, changes in the morphology of the cells due to the shear forces were observed. Offprint requests to: G. Kretzmer     

Influence of rocky substrata on three-dimensional sponge cells model development

Many marine and freshwater organisms are rocky bottom dwellers, and the mineralogical composition of the substratum is known to potentially condition their biology and ecology. In this work, we propose the use of 3D sponge cellular aggregates, called primmorphs, as suitable models for a multidisciplinary study of the mechanisms which regulate the biological responses triggered by the contact with different inorganic substrata. In our experiments, primmorphs obtained from the marine sponge Petrosia ficiformis (Poiret, 1789) were reared on calcium carbonate or on quartzitic substrata, respectively, and their morphological development was described. In parallel, the quantitative expression levels of two genes, silicatein and heat shock protein 70 (HSP70), were evaluated. The first gene is strictly correlated to spiculogenesis and sponge growth, while the second is an important indicator of stress. The results achieved with this in vitro model clearly demonstrate that quartzitic substrata determine the increase of silicatein gene expression, a lower expression of HSP70 gene, and a remarkable difference in primmorphs morphology compared to the analogous samples grown on marble.     

Elevated shear stress protects Escherichia coli cells adhering to surfaces via catch bonds from detachment by soluble inhibitors

Soluble inhibitors find widespread applications as therapeutic drugs to reduce the ability of eukaryotic cells, bacteria, or viruses to adhere to surfaces and host tissues. Mechanical forces resulting from fluid flow are often present under in vivo conditions, and it is commonly presumed that fluid flow will further add to the inhibitive effect seen under static conditions. In striking contrast, we discover that when surface adhesion is mediated by catch bonds, whose bond life increases with increased applied force, shear stress may dramatically increase the ability of bacteria to withstand detachment by soluble competitive inhibitors. This shear stress-induced protection against inhibitor-mediated detachment is shown here for the fimbrial FimH-mannose-mediated surface adhesion of Escherichia coli. Shear stress-enhanced reduction of bacterial detachment has major physiological and therapeutic implications and needs to be considered when developing and screening drugs.     

Influence of fluid shear and microbubbles on bacterial detachment from a surface

Prevention of microbial adhesion and detachment of adhering microorganisms from surfaces is important in many environmental, industrial, and medical applications. Fluid shear is an obvious parameter for stimulating microbial detachment from surfaces, but recently it has been pointed out that a passing air-liquid interface also has potential in stimulating microbial detachment. In the present study, the ability of microbubbles to stimulate detachment of bacterial strains from a glass surface is compared with the effects of fluid flow. Adhesion and detachment of Actinomyces naeslundii T14V-J1, Streptococcus oralis J22, and their coadhering aggregates were studied on glass, mounted in a parallel plate flow chamber. High fluid wall shear rates (11,000 to 16,000 s(-1)) were established in a laminar flow regime in the absence and presence of microbubbles. Wall shear rates stimulated detachment ranging from 70% to 30% for S. oralis and A. naeslundii, respectively. Coadhering aggregates were detached up to 54%. The presence of microbubbles in the flow increased the detachment of A. naeslundii within 2 min of flow from 40% in the absence of microbubbles to 98%, while detachment of neither S. oralis nor coadhering aggregates was affected by the presence of microbubbles. In summary, extremely high fluid flows can be effective in stimulating microbial detachment, depending on the strain involved. The addition of microbubbles to the flow allows the detachment of tenaciously adhering bacteria not detached by flow alone, but not of adhering coaggregates.     

Heterogeneous response of microvascular endothelial cells to shear stress

We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.     

A comparison of thermodynamic approaches to predict the adhesion of dairy microorganisms to solid substrata.

Four different thermodynamic approaches were compared on their usefulness to predict correctly the adhesion of two fouling microogranisms from dairy processing to various solid substrata. The surface free energies of the interacting surfaces were derived from measured contact angles according to: 1. The equation of state; 2. The geometric-mean equation using dispersion and polar components neglecting spreading pressures; 3. The geometric-mean equation using dispersion and polar components while accounting for spreading pressures; and 4. The Lifshitz-van der Waals/Acid-Base approach. All approaches yielded similar surface free energies for the low energy surfaces. Application of approach 1 with different liquids did not give consistent values for the high surface free energy substrata. The dispersion or Lifshiftz-van der Waals components were nearly equal for approaches 2, 3, and 4; however, the polar or acid-base components differed greatly according to the approach followed. Approaches 1 and 2 correctly predicted that adhesion should occur, although the trend with respect to the various solid substrata was opposite the one experimentally observed, as was also the trend predicted by approach 4. Only approach 3 correctly predicted the observed bacterial adhesion with respect to the various solid substrata. In approach 3 and 4, adhesion was frequently found, despite a positive free energy of adhesion. This was attributed to either possible local attractive electrostatic interactions, inadequate weighing of surface free energy components in the calculation of free energies of adhesion, or to additional forces arising from structured interfacial water.     

IL-1beta sensitizes intervertebral disc annulus cells to fluid-induced shear stress.

Chronic inflammation and altered mechanical loading are implicated as contributors to intervertebral disc degeneration. Biomechanical and biochemical factors play a role in disc degeneration but have received limited study. Mechanically, intervertebral discs are sheared during bending or twisting of the trunk. Biochemically, IL-1beta, detected in degenerative discs, promotes metalloproteinase expression. We hypothesized that disc cells might respond to shear stress and IL-1beta in a calcium signaling response. We measured the effect of single and combined stimuli on intracellular calcium concentration ([Ca2+]ic) and signaling. Cells were isolated from annulus tissue, cultured to quiescence, plated on collagen-bonded Culture Slips and incubated with Fura-2AM. Cells then were incubated in IL-1beta. Cell response to the effects of fluid flow was tested using FlexFlo, a laminar flow device. Human annulus (hAN) cells responded to laminar fluid flow with a one to three-fold increase in [Ca2+]ic. IL-1beta alone produced a small, transient stimulation. hAN cells pretreated with IL-1beta responded to shear with a more dramatic and sustained increase in [Ca2+]ic, six to ten-fold over basal level, when compared to shear then IL-1beta or shear and IL-1beta alone (P<0.001 for all comparisons). This is the first study documenting synergism of a signaling response to biomechanical and biochemical stimuli in human disc cells. IL-1beta treatment appeared to "sensitize" annulus cells to mechanical load. This increased responsiveness to mechanical load in the face of inflammatory cytokines may imply that the sensitivity of annulus cells to shear increases during inflammation and may affect initiation and progression of disc degeneration.     

Protective effect of methylcellulose and other polymers on insect cells subjected to laminar shear stress.

The relative sensitivity of two insect cell lines to laminar shear stress was determined, and the protective effect of polymers added to the growth media of two insect cell lines, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), was evaluated. TN-368 and SF-9 cells were found to be equally sensitive to laminar shear stress. Methylcellulose [0.5% (w/v) Dow E4M Methocel] and dextran [4.5% (w/v)] increased the resistance of suspended cells to lysis due to laminar shear stress by factors of up to 76 and 28, respectively, compared to cells in media without additives. It was observed that the protective effect of Pluronic F-68 was concentration-dependent: 0.2% and 0.3% (w/v) F-68 increased the resistance of SF-9 cells to shear stress by factors of 15 and 42, respectively. However, increasing the concentration to 0.5% did not significantly increase the cells' resistance compared to 0.3% (w/v). F-68 at 0.2% only increased the resistance of TN-368 cells by a factor of 6. It is believed that the protection is a result of the polymer adsorbing to the cell membrane. None of the polymer additives tested had a significant effect on SF-9 or TN-368 growth rate.     

Morphological changes and detachment of adherent cells induced by p122, a GTPase-activating protein for Rho.

We recently cloned a novel signaling molecule, p122, that shows a GTPase-activating activity specific for Rho and the ability to enhance the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C delta1 in vitro. Here we analyzed the in vivo function of p122. Microinjection of the GTPase-activating domain of p122 suppressed the formation of stress fibers and focal adhesions induced by lysophosphatidic acid, suggesting a GTPase-activating activity for Rho as in in vitro. Transfection of p122 also induced the disassembly of stress fibers and the morphological rounding of various adherent cells. Analyses using deletion and point mutants demonstrated that the GTPase-activating domain of p122 is responsible for the morphological changes and detachment and that arginine residues at positions 668 and 710 and a lysine residue at position 706 in the GTPase-activating domain are essential. Using Fluo-3-based Ca2+ microscopy, we found that p122 evoked a rapid elevation of intracellular Ca2+ levels, suggesting that p122 stimulates the phosphatidylinositol 4, 5-bisphosphate-hydrolyzing activity of phospholipase C delta1. These results demonstrate that p122 synergistically functions as a GTPase-activating protein specific for Rho and an activator of phospholipase C delta1 in vivo and induces morphological changes and detachment through cytoskeletal reorganization.