Multispecies Bacterial Biofilms and Their Evaluation Using Bioreactors.

Pathogenic biofilm formation within food processing industries raises a serious public health and safety concern, and places burdens on the economy. Biofilm formation on equipment surfaces is a rather complex phenomenon, wherein multiple steps are involved in bacterial biofilm formation. In this review we discuss the stages of biofilm formation, the existing literature on the impact of surface properties and shear stress on biofilms, types of bioreactors, and antimicrobial coatings. The review underscores the significance of prioritizing biofilm prevention strategies as a first line of defense, followed by control measures. Utilizing specific biofilm eradication strategies as opposed to a uniform approach is crucial because biofilms exhibit different behavioral outcomes even amongst the same species when the environmental conditions change. This review is geared towards biofilm researchers and food safety experts, and seeks to derive insights into the scope of biofilm formation, prevention, and control. The use of suitable bioreactors is paramount to understanding the mechanisms of biofilm formation. The findings provide useful information to researchers involved in bioreactor selection for biofilm investigation, and food processors in surfaces with novel antimicrobial coatings, which provide minimal bacterial attachment.

Investigating the effects of membrane deformability on artificial capsule adhesion to the functionalized surface.

Understanding, manipulating and controlling cellular adhesion processes can be critical in developing biomedical technologies. Adhesive mechanisms can be used to the target, pattern and separate cells such as leukocytes from whole blood for biomedical applications. The deformability response of the cell directly affects the rolling and adhesion behavior under viscous linear shear flow conditions. To that end, the primary objective of the present study was to investigate numerically the influence of capsule membrane's nonlinear material behavior (i.e. elastic-plastic to strain hardening) on the rolling and adhesion behavior of representative artificial capsules. Specifically, spherical capsules with radius of [Formula: see text] were represented using an elastic membrane governed by a Mooney-Rivlin strain energy functions. The surfaces of the capsules were coated with P-selectin glycoprotein-ligand-1 to initiate binding interaction with P-selectin-coated planar surface with density of [Formula: see text] under linear shear flow varying from 100 to [Formula: see text]. The numerical model is based on the Immersed Boundary Method for rolling of deformable capsule in shear flow coupled with Monte Carlo simulation for receptor/ligand interaction modeled using Bell model. The results reveal that the mechanical properties of the capsule play an important role in the rolling behavior and the binding kinetics between the capsule contact surface and the substrate. The rolling behavior of the strain hardening capsules is relatively smoother and slower compared to the elastic-plastic capsules. The strain hardening capsules exhibits higher contact area at any given shear rate compared to elastic-plastic capsules. The increase in contact area leads to decrease in rolling velocity. The capsule contact surface is not in complete contact with the substrate because of thin lubrication film that is trapped between the capsule and substrate. This creates a concave shape on the bottom surface of the capsule that is referred to as a dimple. In addition, the present study demonstrates that the average total bond force from the capsules lifetime increases by 37 % for the strain hardening capsules compared to elastic-plastic capsules at shear rate of [Formula: see text]. Finally, the model demonstrates the effect of finite membrane deformation on the coupling between hydrodynamic and receptor/ligand interaction.

Viscoelasticity as a biomarker for high-throughput flow cytometry.

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.

Measuring cell mechanics by optical alignment compression cytometry.

To address the need for a high throughput, non-destructive technique for measuring individual cell mechanical properties, we have developed optical alignment compression (OAC) cytometry. OAC combines hydrodynamic drag in an extensional flow microfluidic device with optical forces created with an inexpensive diode laser to induce measurable deformations between compressed cells. In this, a low-intensity linear optical trap aligns incoming cells with the flow stagnation point allowing hydrodynamic drag to induce deformation during cell-cell interaction. With this novel approach, we measure cell mechanical properties with a throughput that improves significantly on current non-destructive individual cell testing methods.

Erythrocyte deformation in high-throughput optical stretchers.

Optical stretchers can be used to quantify elastic and homeostatic properties of cells. Because they can apply forces to cells without requiring direct contact, they may noninvasively measure mechanical properties related to cell and membrane health. Present-day optical stretchers are, however, limited to measurements on individual stationary cells, limiting throughput. To overcome this limitation and allow study of variations in cell populations, we recently developed and tested a microfluidic chamber that measures optical stretching parameters for erythrocytes under dynamic flowing conditions. The method uses a single linear diode laser bar and permitted measurements at low flow rates and higher throughput. Here, we numerically investigate the feasibility of further increasing the measurement rates of the optical stretcher in parameter domains where hydrodynamic and optical forces are of comparable magnitude. To do this we couple a recently implemented dynamic optical ray-tracing technique with a fluid-structure interaction solver to simulate the deformation of osmotically swollen erythrocytes in fluid flow of variable rate. Our results demonstrate that a detectable steady-state stretch is induced at nominal optical powers and flow rates. In addition, we find that flow rates can be increased significantly with no major effect on net cell stretch showing the feasibility of application of this technique at greatly increased throughputs.

Elastic capsule deformation in general irrotational linear flows.

Knowledge of the response of elastic capsules to imposed fluid flow is necessary for predicting deformation and motion of biological cells and synthetic capsules in microfluidic devices and in the microcirculation. Capsules have been studied in shear, planar extensional, and axisymmetric extensional flows. Here, the flow gradient matrix of a general irrotational linear flow is characterized by two parameters, its strain rate, defined as the maximum of the principal strain rates, and by a new term, q, the difference in the two lesser principal strain rates, scaled by the maximum principal strain rate; this characterization is valid for ellipsoids in irrotational linear flow, and it gives good results for spheres in general linear flows at low capillary numbers. We demonstrate that deformable non-spherical particles align with the principal axes of an imposed irrotational flow. Thus, it is most practical to model deformation of non-spherical particles already aligned with the flow, rather than considering each arbitrary orientation. Capsule deformation was modeled for a sphere, a prolate spheroid, and an oblate spheroid, subjected to combinations of uniaxial, biaxial, and planar extensional flows; modeling was performed using the immersed boundary method. The time response of each capsule to each flow was found, as were the steady-state deformation factor, mean strain energy, and surface area. For a given capillary number, planar flows led to more deformation than uniaxial or biaxial extensional flows. Capsule behavior in all cases was bounded by the response of capsules to uniaxial, biaxial, and planar extensional flow.

Dynamic ray tracing for modeling optical cell manipulation.

Current methods for predicting stress distribution on a cell surface due to optical trapping forces are based on a traditional ray optics scheme for fixed geometries. Cells are typically modeled as solid spheres as this facilitates optical force calculation. Under such applied forces however, real and non-rigid cells can deform, so assumptions inherent in traditional ray optics methods begin to break down. In this work, we implement a dynamic ray tracing technique to calculate the stress distribution on a deformable cell induced by optical trapping. Here, cells are modeled as three-dimensional elastic capsules with a discretized surface with associated hydrodynamic forces calculated using the Immersed Boundary Method. We use this approach to simulate the transient deformation of spherical, ellipsoidal and biconcave capsules due to external optical forces induced by a single diode bar optical trap for a range of optical powers.

Cell deformation cytometry using diode-bar optical stretchers.

The measurement of cell elastic parameters using optical forces has great potential as a reagent-free method for cell classification, identification of phenotype, and detection of disease; however, the low throughput associated with the sequential isolation and probing of individual cells has significantly limited its utility and application. We demonstrate a single-beam, high-throughput method where optical forces are applied anisotropically to stretch swollen erythrocytes in microfluidic flow. We also present numerical simulations of model spherical elastic cells subjected to optical forces and show that dual, opposing optical traps are not required and that even a single linear trap can induce cell stretching, greatly simplifying experimental implementation. Last, we demonstrate how the elastic modulus of the cell can be determined from experimental measurements of the equilibrium deformation. This new optical approach has the potential to be readily integrated with other cytometric technologies and, with the capability of measuring cell populations, enabling true mechanical-property-based cell cytometry.

Multi-scale simulation of L-selectin-PSGL-1-dependent homotypic leukocyte binding and rupture.

L-selectin-PSGL-1-mediated polymorphonuclear (PMN) leukocyte homotypic interactions potentiate the extent of PMN recruitment to endothelial sites of inflammation. Cell-cell adhesion is a complex phenomenon involving the interplay of bond kinetics and hydrodynamics. As a first step, a 3-D computational model based on the Immersed Boundary Method is developed to simulate adhesion-detachment of two PMN cells in quiescent conditions. Our simulations predict that the total number of bonds formed is dictated by the number of available receptors (PSGL-1) when ligands (L-selectin) are in excess, while the excess amount of ligands influences the rate of bond formation. Increasing equilibrium bond length results in a higher number of receptor-ligand bonds due to an increased intercellular contact area. On-rate constants determine the rate of bond formation, while off-rates control the average number of bonds by modulating bond lifetimes. Application of an external pulling force leads to time-dependent on- and off-rates and causes bond rupture. Moreover, the time required for bond rupture in response to an external force is inversely proportional to the applied load and decreases with increasing off-rate.

Linear diode laser bar optical stretchers for cell deformation.

To investigate the use of linear diode laser bars to optically stretch cells and measure their mechanical properties, we present numerical simulations using the immersed boundary method (IBM) coupled with classic ray optics. Cells are considered as three-dimensional (3D) spherical elastic capsules immersed in a fluid subjected to both optical and hydrodynamic forces in a periodic domain. We simulate cell deformation induced by both single and dual diode laser bar configurations and show that a single diode laser bar induces significant stretching but also induces cell translation of speed < 10 µm/sec for applied 6.6 mW/µm power in unconfined systems. The dual diode laser bar configuration, however, can be used to both stretch and optically trap cells at a fixed position. The net cell deformation was found to be a function of the total laser power and not the power distribution between single or dual diode laser bar configurations.

Effects of a surfactant monolayer on the measurement of equilibrium interfacial tension of a drop in extensional flow.

The effect of surfactant monolayer concentration on the measurement of interfacial surface tension using transient drop deformation methods is studied using the Boundary Integral Method. Emulsion droplets with a surfactant monolayer modeled with the Langmuir equation of state initially in equilibrium are suddenly subjected to axisymmetric extensional flows until a steady state deformation is reached. The external flow is then removed and the retraction of the drops to a spherical equilibrium shape in a quiescent state is simulated. The transient response of the drop to the imposed flow is analyzed to obtain a characteristic response time, tau(s)( *). Neglecting the initial and final stages, the retraction process can be closely approximated by an exponential decay with a characteristic time, tau(r)( *). The strength of the external flow on each model drop is increased in order to investigate the coupled effect of deformation and surfactant distribution on the characteristic relaxation time. Different model drops are considered by varying the internal viscosity and the equilibrium surfactant concentrations from a surfactant free state (clean) to high concentrations approaching the maximum packing limit. The characteristic times obtained from the simulated drop dynamics both in extension and retraction are used to determine an apparent surface tension employing linear theory. In extension the apparent surface tension under predicts the prescribed equilibrium surface tension. The error increases monotonically with the equilibrium surfactant concentration and diverges as the maximum packing limit is approached. In retraction the apparent surface tension under predicts the prescribed equilibrium surface tension depends non-monotonically on the equilibrium surfactant concentration. The error is highest for moderate surfactant concentrations and decreases as the maximum packing limit is approached. It was found that the difference between the prescribed surface tension and the apparent surface tension increased as the viscosity ratio decreased. Differences as large as 40% were seen between the prescribed surface tension and the apparent surface tension predicted by the linear theory.

Roles of cell and microvillus deformation and receptor-ligand binding kinetics in cell rolling.

Polymorphonuclear leukocyte (PMN) recruitment to sites of inflammation is initiated by selectin-mediated PMN tethering and rolling on activated endothelium under flow. Cell rolling is modulated by bulk cell deformation (mesoscale), microvillus deformability (microscale), and receptor-ligand binding kinetics (nanoscale). Selectin-ligand bonds exhibit a catch-slip bond behavior, and their dissociation is governed not only by the force but also by the force history. Whereas previous theoretical models have studied the significance of these three "length scales" in isolation, how their interplay affects cell rolling has yet to be resolved. We therefore developed a three-dimensional computational model that integrates the aforementioned length scales to delineate their relative contributions to PMN rolling. Our simulations predict that the catch-slip bond behavior and to a lesser extent bulk cell deformation are responsible for the shear threshold phenomenon. Cells bearing deformable rather than rigid microvilli roll slower only at high P-selectin site densities and elevated levels of shear (>or=400 s(-1)). The more compliant cells (membrane stiffness=1.2 dyn/cm) rolled slower than cells with a membrane stiffness of 3.0 dyn/cm at shear rates >50 s(-1). In summary, our model demonstrates that cell rolling over a ligand-coated surface is a highly coordinated process characterized by a complex interplay between forces acting on three distinct length scales.

Mathematical modeling of cell adhesion in shear flow: application to targeted drug delivery in inflammation and cancer metastasis.

Cell adhesion plays a pivotal role in diverse biological processes that occur in the dynamic setting of the vasculature, including inflammation and cancer metastasis. Although complex, the naturally occurring processes that have evolved to allow for cell adhesion in the vasculature can be exploited to direct drug carriers to targeted cells and tissues. Fluid (blood) flow influences cell adhesion at the mesoscale by affecting the mechanical response of cell membrane, the intercellular contact area and collisional frequency, and at the nanoscale level by modulating the kinetics and mechanics of receptor-ligand interactions. Consequently, elucidating the molecular and biophysical nature of cell adhesion requires a multidisciplinary approach involving the synthesis of fundamentals from hydrodynamic flow, molecular kinetics and cell mechanics with biochemistry/molecular cell biology. To date, significant advances have been made in the identification and characterization of the critical cell adhesion molecules involved in inflammatory disorders, and, to a lesser degree, in cancer metastasis. Experimental work at the nanoscale level to determine the lifetime, interaction distance and strain responses of adhesion receptor-ligand bonds has been spurred by the advent of atomic force microscopy and biomolecular force probes, although our current knowledge in this area is far from complete. Micropipette aspiration assays along with theoretical frameworks have provided vital information on cell mechanics. Progress in each of the aforementioned research areas is key to the development of mathematical models of cell adhesion that incorporate the appropriate biological, kinetic and mechanical parameters that would lead to reliable qualitative and quantitative predictions. These multiscale mathematical models can be employed to predict optimal drug carrier-cell binding through isolated parameter studies and engineering optimization schemes, which will be essential for developing effective drug carriers for delivery of therapeutic agents to afflicted sites of the host.

Shear modulation of intercellular contact area between two deformable cells colliding under flow.

Shear rate has been shown to critically affect the kinetics and receptor specificity of cell-cell interactions. In this study, the collision process between two modeled cells interacting in a linear shear flow is numerically investigated. The two identical biological or artificial cells are modeled as deformable capsules composed of an elastic membrane. The cell deformation and trajectories are computed using the immersed boundary method (IBM) for shear rates of 100-400s(-1). As the two cells collide under hydrodynamic shear, large local cell deformations develop. The effective contact area between the two cells is modulated by the shear rate, and reaches a maximum value at intermediate levels of shear. At relatively low shear rate, the contact area is an enclosed region. As the shear rate increases, dimples form on the membrane surface, and the contact region becomes annular. The nonmonotonic increase of the contact area with the increase of shear rate from computational results implies that there is a maximum effective receptor-ligand binding area for cell adhesion. This finding suggests the existence of possible hydrodynamic mechanism that could be used to interpret the observed maximum leukocyte aggregation in shear flow. The critical shear rate for maximum intercellular contact area is shown to vary with cell properties such as radius and membrane elastic modulus.

A 3-D computational model predicts that cell deformation affects selectin-mediated leukocyte rolling.

Leukocyte recruitment to sites of inflammation is initiated by their tethering and rolling on the activated endothelium under flow. Even though the fast kinetics and high tensile strength of selectin-ligand bonds are primarily responsible for leukocyte rolling, experimental evidence suggests that cellular properties such as cell deformability and microvillus elasticity actively modulate leukocyte rolling behavior. Previous theoretical models either assumed cells as rigid spheres or were limited to two-dimensional representations of deformable cells with deterministic receptor-ligand kinetics, thereby failing to accurately predict leukocyte rolling. We therefore developed a three-dimensional computational model based on the immersed boundary method to predict receptor-mediated rolling of deformable cells in shear flow coupled to a Monte Carlo method simulating the stochastic receptor-ligand interactions. Our model predicts for the first time that the rolling of more compliant cells is relatively smoother and slower compared to cells with stiffer membranes, due to increased cell-substrate contact area. At the molecular level, we show that the average number of bonds per cell as well as per single microvillus decreases with increasing membrane stiffness. Moreover, the average bond lifetime decreases with increasing shear rate and with increasing membrane stiffness, due to higher hydrodynamic force experienced by the cell. Taken together, our model captures the effect of cellular properties on the coupling between hydrodynamic and receptor-ligand bond forces, and successfully explains the stable leukocyte rolling at a wide range of shear rates over that of rigid microspheres.

Viscosity of animal erythrocyte suspensions mixed with a perflurocarbon emulsion.

Artificial blood substitutes (ABS) offer an alternative to donated blood. Increasing the oxygen carrying capacity of blood plasma through the addition of a Perfluorocarbon emulsion (PFE) is one approach in creating a blood substitute. The peripheral resistance of the circulatory system may be altered depending on the rheological properties of the ABS. Measurements of the rheological behavior of mixtures of a PFE, Oxygent, and erythrocyte suspensions were conducted at room temperature at different hematocrits using sheep (nonaggregating) and swine (aggregating) erythrocytes. The pure PFE was found to be shear thinning. Adding 6 and 12 g per 100 mL of sheep blood at the various hematocrits increased the viscosity of the suspension from as low as 4% (6 g PFE/40% Hct) to as high as 26.5% (12 g PFE/plasma). The nonaggregating sheep erythrocyte and PFE mixtures exhibited Newtonian behavior. Shear thinning behavior continued upon addition of 6 and 12 g per 100 mL of swine blood at the various hematocrits, with a slight increase in viscosity in most cases. It was observed that adding 12 g of PFE (approximately 3 x intended clinical dose) to 40% Hct swine blood at room temperature led to a significant decrease in viscosity.

Using computational fluid dynamics software to estimate circulation time distributions in bioreactors.

Nonideal mixing in many fermentation processes can lead to concentration gradients in nutrients, oxygen, and pH, among others. These gradients are likely to influence cellular behavior, growth, or yield of the fermentation process. Frequency of exposure to these gradients can be defined by the circulation time distribution (CTD). There are few examples of CTDs in the literature, and experimental determination of CTD is at best a challenging task. The goal in this study was to determine whether computational fluid dynamics (CFD) software (FLUENT 4 and MixSim) could be used to characterize the CTD in a single-impeller mixing tank. To accomplish this, CFD software was used to simulate flow fields in three different mixing tanks by meshing the tanks with a grid of elements and solving the Navier-Stokes equations using the kappa-epsilon turbulence model. Tracer particles were released from a reference zone within the simulated flow fields, particle trajectories were simulated for 30 s, and the time taken for these tracer particles to return to the reference zone was calculated. CTDs determined by experimental measurement, which showed distinct features (log-normal, bimodal, and unimodal), were compared with CTDs determined using CFD simulation. Reproducing the signal processing procedures used in each of the experiments, CFD simulations captured the characteristic features of the experimentally measured CTDs. The CFD data suggests new signal processing procedures that predict unimodal CTDs for all three tanks.

Fluid shear contributions to bacteria cell detachment initiated by a monoclonal antibody.

Receptor-mediated adhesion of bacteria to biological surfaces is a significant step leading to infection. Due to an increase in bacterial antibiotic resistance, novel methods to block and disrupt these specific interactions have gained considerable interest as possible therapeutic strategies. Recently, several monoclonal antibodies specific for the Staphylococcus aureus collagen receptor demonstrated specialized ability to displace attached cells from collagen in static assays. In this study, we experimentally examine the monoclonal antibody detachment functionality under physiological shear conditions to evaluate the role of this parameter in the detachment process. The detachment of staphylococci from collagen was quantified in real-time using a parallel plate flow chamber, phase contrast video-microscopy and digital image processing. The results demonstrate a unimodal dependence of detachment on fluid wall shear rate. The observed decrease in effective detachment rate with increasing force at the highest shear levels evaluated is counterintuitive and has not been previously demonstrated. Several possible mechanisms of this result are discussed.