Induction of mammalian cell death by simple shear and extensional flows

In this work we investigated whether the type of shear flow, to which cells are exposed, influences the initiation of cell death. It is shown that mammalian cells, indeed, distinguish between discrete types of flow and respond differently. Two flow devices were employed to impose accurate hydrodynamic flow fields: uniform steady simple shear flow and oscillating extensional flow. To distinguish between necrotic and apoptotic cell death, fluorescence activated cell sorting and the release of DNA in the culture supernatant was used. Results show that Chinese Hamster Ovaries and Human Embryonic Kidney cells will enter the apoptotic pathway when subjected to low levels of hydrodynamic stress (around 2.0 Pa) in oscillating, extensional flow. In contrast, necrotic death prevails when the cells are exposed to hydrodynamic stresses around 1.0 Pa in simple shear flow or around 500 Pa in extensional flow. These threshold values at which cells enter the respective death pathway should be avoided when culturing cells for recombinant protein production to enhance culture longevity and productivity. Biotechnol. Bioeng. 2009; 104: 360–370 © 2009 Wiley Periodicals, Inc.     

Critical stresses for cancer cell detachment in microchannels

We present experiments involving cancer cells adhering to microchannels, subjected to increasing shear stresses (0.1–30 Pa). Morphological studies were carried out at different shear stresses. Cells exhibit spreading patterns similar to those observed under static conditions, as long as the shear stress is not too high. At critical wall shear stresses (around 2−5 Pa), cell-substrate contact area decreases until detachment at the larger stresses. Critical shear stresses are found to be lower for higher confinements (i.e. smaller cell height to channel height ratio). Fluorescent techniques were used to locate focal adhesions (typically 1 μm² in size) under various shearing conditions, showing that cells increase the number of focal contacts in the region facing the flow. To analyze such data, we propose a model to determine the critical stress, resulting from the competition between hydrodynamic forces and the adhesive cell resistance. With this model, typical adhesive stresses exerted at each focal contact can be determined and are in agreement with previous works.     

Effects of shear flow on photosynthesis in a dilute suspension of microalgae

This study investigates the effects of shear stress on photosynthesis in dilute suspensions of Spirulina platensis and Chlorella by measuring the oxygen production rate using a coaxial, double-rotating-cylinder apparatus that generates Couette shear flow. Our device enables up to 0.6 Pa shear stress to be applied, which has the hydrodynamic effect of generating the algal motion and acutely augmenting the oxygen production rate of Spirulina, primarily because the surface area of algae exposed to illumination is increased. However, there is shear-flow limitation on any increase in oxygen production, and the shear stress at maximum oxygen production rate tends to decrease with increasing temperature. The comparative study with Chlorella showed the reverse relationship between oxygen production and shear stress, and the cause of this difference is discussed in terms of several factors such as size, shape, hydrodynamic stress capacity and others.     

Removal kinetics of Bacillus cereus spores from a stainless steel surface exposed to constant shear stress 1 ‐ experimental system

The efficiency of cleaning in place procedures in dairy industries can be greatly affected by the presence of spore‐forming bacteria, which are able to adhere strongly to surfaces and to survive disinfection procedures. Microbial adhesion has been extensively studied, but very few studies have yet reported on the hydrodynamic removal of microorganisms, due to the lack of simple, routinely performable techniques. In this paper, a methodology using a coaxial cylinder double gap viscometer is described, to study the removal kinetics of Bacillus cereus spores from a stainless steel support under hydrodynamic conditions. This method was shown to be reproducible, sensitive and easy to perform, and allowed spore hydrodynamic removal kinetics to be studied as a function of both adhesion and detachment conditions. A high ionic strength attachment medium was shown to enhance adhesion forces, provided it did not contain macromolecules. An increase in shear stress was found to be favorable to spore detachment (4 to 5 times more spores were detached at 28 Pa than at 2 Pa), but removal kinetics were not found to be significantly different for 2 and 15 Pa. Thus, the effect of shear stress on spore removal kinetics may not be linear.     

Effect of oscillating fluid flow stimulation on osteocyte mRNA expression

Structural adaptation of the bone tissue is mediated by loading-induced interstitial fluid flow within the bone microstructure. Within this framework, osteocytes fulfill the central mechanotransductive role in the bone remodeling process. While osteocytes have been demonstrated to be exquisitely sensitive to various forms of fluid flow stimulus in vitro, the effect of different oscillating fluid flow (OFF) parameters on osteocyte activity has yet to be systematically characterized. In this study, we investigate the effect of three OFF parameters on osteocyte activity in vitro and hypothesize that COX-2, RANKL, and OPG mRNA expression in osteocytes are sensitive to the OFF parameters: peak shear stress amplitude (0.5 Pa, 1 Pa, 2 Pa, and 5 Pa), oscillating frequency (0.5 Hz, 1 Hz, and 2 Hz), and total flow duration (1 h, 2 h, and 4 h). Our findings demonstrate that COX-2 mRNA levels are elevated in osteocytes subjected to higher peak shear stress amplitudes and longer flow durations, while RANKL/OPG mRNA levels decreased to a minimum threshold in response to higher peak shear stress amplitudes, faster oscillating frequencies, and longer flow durations. These findings suggest that dynamic fluid flow with higher peak shear stress amplitudes, faster oscillating frequencies, and longer loading durations provide the best conditions for promoting bone formation.     

Flow cell hydrodynamics and their effects on E. coli biofilm formation under different nutrient conditions and turbulent flow

Biofilm formation is a major factor in the growth and spread of both desirable and undesirable bacteria as well as in fouling and corrosion. In order to simulate biofilm formation in industrial settings a flow cell system coupled to a recirculating tank was used to study the effect of a high (550 mg glucose l?1) and a low (150 mg glucose l?1) nutrient concentration on the relative growth of planktonic and attached biofilm cells of Escherichia coli JM109(DE3). Biofilms were obtained under turbulent flow (a Reynolds number of 6000) and the hydrodynamic conditions of the flow cell were simulated by using computational fluid dynamics. Under these conditions, the flow cell was subjected to wall shear stresses of 0.6 Pa and an average flow velocity of 0.4 m s?1 was reached. The system was validated by studying flow development on the flow cell and the applicability of chemostat model assumptions. Full development of the flow was assessed by analysis of velocity profiles and by monitoring the maximum and average wall shear stresses. The validity of the chemostat model assumptions was performed through residence time analysis and identification of biofilm forming areas. These latter results were obtained through wall shear stress analysis of the system and also by assessment of the free energy of interaction between E. coli and the surfaces. The results show that when the system was fed with a high nutrient concentration, planktonic cell growth was favored. Additionally, the results confirm that biofilms adapt their architecture in order to cope with the hydrodynamic conditions and nutrient availability. These results suggest that until a certain thickness was reached nutrient availability dictated biofilm architecture but when that critical thickness was exceeded mechanical resistance to shear stress (ie biofilm cohesion) became more important.     

Minimizing hydrodynamic stress in mammalian cell culture through the lobed Taylor-Couette bioreactor

The objective of the present study was to investigate the effect of hydrodynamic stress heterogeneity on metabolism and productivity of an industrial mammalian cell line. For this purpose, a novel Lobed Taylor-Couette (LTC) mixing unit combining a narrow distribution of hydrodynamic stresses and a membrane aeration system to prevent cell damage by bubble bursting was developed. A hydrodynamic analysis of the LTC was developed to reproduce, in a uniform hydrodynamic environment, the same hydrodynamic stress encountered locally by cells in a stirred tank, particularly at the large scale, e.g., close and far from the impeller. The developed LTC was used to simulate the stress values near the impeller of a laboratory stirred tank bioreactor, equal to about 0.4 Pa, which is however below the threshold value leading to cell death. It was found that the cells actively change their metabolism by increasing lactate production and decreasing titer while the consumption of the main nutrients remains substantially unchanged. When considering average stress values ranging from 1 to 10 Pa found by other researchers to cause physiological response of cells to the hydrodynamic stress in heterogeneous stirred vessels, our results are close to the lower boundary of this interval.     

Osteoblasts respond to pulsatile fluid flow with short-term increases in PGE(2) but no change in mineralization.

Although there is no consensus as to the precise nature of the mechanostimulatory signals imparted to the bone cells during remodeling, it has been postulated that deformation-induced fluid flow plays a role in the mechanotransduction pathway. In vitro, osteoblasts respond to fluid shear stress with an increase in PGE(2) production; however, the long-term effects of fluid shear stress on cell proliferation and differentiation have not been examined. The goal of this study was to apply continuous pulsatile fluid shear stresses to osteoblasts and determine whether the initial production of PGE(2) is associated with long-term biochemical changes. The acute response of bone cells to a pulsatile fluid shear stress (0.6 +/- 0.5 Pa, 3.0 Hz) was characterized by a transient fourfold increase in PGE(2) production. After 7 days of static culture (0 dyn/cm(2)) or low (0.06 +/- 0.05 Pa, 0.3 Hz) or high (0.6 +/- 0.5 Pa, 3.0 Hz) levels of pulsatile fluid shear stress, the bone cells responded with an 83% average increase in cell number, but no statistical difference (P > 0.53) between the groups was observed. Alkaline phosphatase activity per cell decreased in the static cultures but not in the low- or high-flow groups. Mineralization was also unaffected by the different levels of applied shear stress. Our results indicate that short-term changes in PGE(2) levels caused by pulsatile fluid flow are not associated with long-term changes in proliferation or mineralization of bone cells.     

Influence of hydrodynamic stress on the frictional drag of biofouling communities

The role of hydrodynamic wall shear stresses on the development of the fouling community structure and resulting frictional drag were examined using a commercially available fouling release coating. Immersed test panels were exposed to three different hydrodynamic treatments, one static and two dynamic (corresponding to an estimated wall shear stress of 7.0 and 25.5 Pa). The drag of the panels was measured in a hydrodynamic test chamber at discrete time intervals over 35 days. The fouling community composition on the static panels was significantly different from the organisms observed on the dynamic panels. Despite different fouling community composition, the drag forces measured on the panels were very similar. This suggests that the frictional drag of low form and soft fouling communities are similar and that there may be a stepwise increase in frictional drag associated with the presence of mature calcareous organisms.     

Dielectric spectroscopy as a novel and convenient tool for the study of the shear sensitivity of plant cells in suspension culture.

Plant cell suspensions of different species and different age were subjected to hydrodynamic stress while following the decline in the volume fraction of intact cells by measuring the permittivity of the cell suspension at radio frequencies. Results were compared with the fresh weight, dry weight, packed cell volume and cell number of the suspensions. At first a rapid decline is seen as the most shear-sensitive cells are broken up, followed by a slower decline as less sensitive cells are broken up. The sensitivity of the cells to shear stress depended strongly on the cell line used but only slightly on their age, older cells being more sensitive. The dependence of the shear sensitivity on the cell line might be an effect of the species investigated, the culturing conditions of the cell line, or both. It was found that cells that grow in a finely dispersed suspension are much less prone to shear stress than is often assumed.     

Evidence for shear-induced increase in membrane fluidity in the dinoflagellate <Emphasis Type="Italic">Lingulodinium polyedrum</Emphasis>

Fluid shear stress has been demonstrated to affect the structure and function of various cell types. In mammalian cells, it was hypothesized that shear-induced membrane fluidization leads to activation of heterotrimetric G-proteins. The purpose of this study was to determine if a similar mechanism exists in the dinoflagellate Lingulodinium polyedrum, a single-celled eukaryotic aquatic organism that bioluminesces under shear stress. Membrane fluidity changes in L. polyedrum were monitored using the molecular rotor 9-(dicyanovinyl)-julolidine, whose fluorescence intensity changes inversely with membrane fluidity. Dual-staining with 9-(dicyanovinyl)-julolidine and the membrane dye 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate indicates membrane localization. Subjecting L. polyedrum cells to increasing shear stress reversibly decreased 9-(dicyanovinyl)-julolidine fluorescence, while autofluorescence of the cytoplasmic chlorophyll did not change. The relationship between shear stress (0.63 Pa, 1.25 Pa, 1.88 Pa, and 2.5 Pa) and membrane fluidity changes was linear and dose-dependent with a 12% increase in fluidity at 2.5 Pa. To further explore this mechanism a membrane fluidizing agent, dimethyl sulfoxide was added. Dimethyl sulfoxide decreased 9-(dicyanovinyl)-julolidine emission by 41+/-15% and elicited a dose-dependent bioluminescent response at concentrations of 0.2%, 0.5%, 1.0%, and 1.25%. This study demonstrates a link between fluid shear stress and membrane fluidity, and suggests that the membrane is an important flow mechanosensor of dinoflagellates.     

Optimizing the rotor design for controlled-shear affinity filtration using computational fluid dynamics

Controlled shear affinity filtration (CSAF) is a novel integrated processing technology that positions a rotor directly above an affinity membrane chromatography column to permit protein capture and purification directly from cell culture. The conical rotor is intended to provide a uniform and tunable shear stress at the membrane surface that inhibits membrane fouling and cell cake formation by providing a hydrodynamic force away from and a drag force parallel to the membrane surface. Computational fluid dynamics (CFD) simulations are used to show that the rotor in the original CSAF device (Vogel et al., 2002) does not provide uniform shear stress at the membrane surface. This results in the need to operate the system at unnecessarily high rotor speeds to reach a required shear stress of at least 0.17 Pa at every radial position of the membrane surface, compromising the scale-up of the technology. Results from CFD simulations are compared with particle image velocimetry (PIV) experiments and a numerical solution for low Reynolds number conditions to confirm that our CFD model accurately describes the hydrodynamics in the rotor chamber of the CSAF device over a range of rotor velocities, filtrate fluxes, and (both laminar and turbulent) retentate flows. CFD simulations were then carried out in combination with a root-finding method to optimize the shape of the CSAF rotor. The optimized rotor geometry produces a nearly constant shear stress of 0.17 Pa at a rotational velocity of 250 rpm, 60% lower than the original CSAF design. This permits the optimized CSAF device to be scaled up to a maximum rotor diameter 2.5 times larger than is permissible in the original device, thereby providing more than a sixfold increase in volumetric throughput.     

Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells

The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.     

Mechanical Loading by Fluid Shear Stress of Myotube Glycocalyx Stimulates Growth Factor Expression and Nitric Oxide Production

Skeletal muscle fibers have the ability to increase their size in response to a mechanical overload. Finite element modeling data suggest that mechanically loaded muscles in vivo may experience not only tensile strain but also shear stress. However, whether shear stress affects biological pathways involved in muscle fiber size adaptation in response to mechanical loading is unknown. Therefore, our aim was twofold: (1) to determine whether shear stress affects growth factor expression and nitric oxide (NO) production by myotubes, and (2) to explore the mechanism by which shear stress may affect myotubes in vitro. C2C12 myotubes were subjected to a laminar pulsating fluid flow (PFF; mean shear stress 0.4, 0.7 or 1.4 Pa, 1 Hz) or subjected to uni-axial cyclic strain (CS; 15 % strain, 1 Hz) for 1 h. NO production during 1-h PFF or CS treatment was quantified using Griess reagent. The glycocalyx was degraded using hyaluronidase, and stretch-activated ion channels (SACs) were blocked using GdCl₃. Gene expression was analyzed immediately after 1-h PFF (1.4 Pa, 1 Hz) and at 6 h post-PFF treatment. PFF increased IGF-I Ea, MGF, VEGF, IL-6, and COX-2 mRNA, but decreased myostatin mRNA expression. Shear stress enhanced NO production in a dose-dependent manner, while CS induced no quantifiable increase in NO production. Glycocalyx degradation and blocking of SACs ablated the shear stress-stimulated NO production. In conclusion, shear stress activates signaling pathways involved in muscle fiber size adaptation in myotubes, likely via membrane-bound mechanoreceptors. These results suggest that shear stress exerted on myofiber extracellular matrix plays an important role in mechanotransduction in muscle.     

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.     

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When the liver is damaged, hepatic stellate cells (HSCs) can change into an activated, highly migratory state. The migration of HSCs may be affected by shear stress due not only to sinusoidal flow but also by the flow in the space of Disse because this space is filled with blood plasma. In this study, we evaluated the effects of shear stress on HSC migration in a scratch-wound assay with a parallel flow chamber. At regions upstream of the wound area, the migration was inhibited by 0.6 Pa and promoted by 2.0 Pa shear stress, compared to the static condition. The platelet-derived growth factor (PDGF)-BB receptor, PDGFR-β, was expressed in all conditions and the differences were not significant. PDGF increased HSC migration, except at 0.6 Pa shear stress, which was still inhibited. These results indicate that another molecular factor, such as PDGFR-α, may act to inhibit the migration under low shear stress. At regions downstream of the wound area, the migration was smaller under shear stress than under the static condition, although the expression of PDGFR-β was significantly higher. In particular, the migration direction was opposite to the wound area under high shear stress; therefore, migration might be influenced by the intercellular environment. Our results indicate that HSC migration was influenced by shear stress intensity and the intercellular environment.     

Pulsatile extracorporeal circulation during on-pump cardiac surgery enhances aortic wall shear stress

Controversy on superiority of pulsatile versus non-pulsatile extracorporeal circulation in cardiac surgery still continues. Stroke as one of the major adverse events during cardiopulmonary bypass is, in the majority of cases, caused by mobilization of aortic arteriosclerotic plaques that is inducible by pathologically elevated wall shear stress values. The present study employs computational fluid dynamics to evaluate the aortic blood flow and wall shear stress profiles under the influence of antegrade or retrograde perfusion with pulsatile versus non-pulsatile extracorporeal circulation. While, compared to physiological flow, a non-pulsatile perfusion resulted in generally decreased blood velocities and only moderately increased shear forces (48 Pa versus 20 Pa antegradely and 127 Pa versus 30 Pa retrogradely), a pulsatile perfusion extensively enhanced the occurrence of turbulences, maximum blood flow speed and maximum wall shear stress (1020 Pa versus 20 Pa antegradely and 1178 Pa versus 30 Pa retrogradely). Under these circumstances arteriosclerotic embolism has to be considered. Further simulations and experimental work are necessary to elucidate the impact of our findings on the scientific discourse of pulsatile versus non-pulsatile extracorporeal circulation.     

Fluid shear stress in trabecular bone marrow due to low-magnitude high-frequency vibration

Low-magnitude high-frequency (LMHF) loading has recently received attention for its anabolic effect on bone. The mechanism of transmission of the anabolic signal is not fully understood, but evidence indicates that it is not dependent on bone matrix strain. One possible source of signaling is mechanostimulation of the cells in the bone marrow. We hypothesized that the magnitude of the fluid shear stress in the marrow during LMHF loading is in the mechanostimulatory range. As such, the goal of this study was to determine the range of shear stress in the marrow during LMHF vibration. The shear stress was estimated from computational models, and its dependence on bone density, architecture, permeability, marrow viscosity, vibration amplitude and vibration frequency were examined. Three-dimensional finite element models of five trabecular bone samples from different anatomic sites were constructed, and a sinusoidal velocity profile was applied to the models. In human bone models during axial vibration at an amplitude of 1 g, more than 75% of the marrow experienced shear stress greater than 0.5Pa. In comparison, in vitro studies indicate that fluid induced shear stress in the range of 0.5 to 2.0Pa is anabolic to a variety of cells in the marrow. Shear stress at the bone-marrow interface was as high as 5.0Pa. Thus, osteoblasts and bone lining cells that are thought to reside on the endosteal surfaces may experience very high shear stress during LMHF loading. However, a more complete understanding of the location of the various cell populations in the marrow is needed to quantify the effects on specific cell types. This study suggests the shear stress within bone marrow in real trabecular architecture during LMHF vibration could provide the mechanical signal to marrow cells that leads to bone anabolism.     

Microscopic investigation of erythrocyte deformation dynamics

The understanding of erythrocyte deformation under conditions of high shear stress and short exposure time is central to the study of hemorheology and hemolysis within prosthetic blood contacting devices. A combined computational and experimental microscopic study was conducted to investigate the erythrocyte deformation and its relation to transient stress fields. A microfluidic channel system with small channels fabricated using polydimethylsiloxane on the order of 100 mum was designed to generate transient stress fields through which the erythrocytes were forced to flow. The shear stress fields were analyzed by three-dimensional computational fluid dynamics. Microscopic images of deforming erythrocytes were experimentally recorded to obtain the changes in cell morphology over a wide range of fluid dynamic stresses. The erythrocyte elongation index (EI) increased from 0 to 0.54 with increasing shear stress up to 123 Pa. In this shear stress range, erythrocytes behaved like fluid droplets, and deformed and flowed following the surrounding fluid. Cells exposed to shear stress beyond 123 Pa (up to 5170 Pa) did not exhibit additional elongation beyond EI=0.54. Two-stage deformation of erythrocytes in response to shear stress was observed: an initial linear elongation with increasing shear stress and a plateau beyond a critical shear stress.