Experimental characterization of flow conditions in 2- and 20-L bioreactors with wave-induced motion

Quantifying the influence of flow conditions on cell viability is essential for a successful control of cell growth and cell damage in major biotechnological applications, such as in recombinant protein and antibody production or vaccine manufacturing. In the last decade, new bioreactor types have been developed. In particular, bioreactors with wave‐induced motion show interesting properties (e.g., disposable bags suitable for cGMP manufacturing, no requirement for cleaning and sterilization of cultivation vessels, and fast setup of new production lines) and are considered in this study. As an additional advantage, it is expected that cultivations in such bioreactors result in lower shear stress compared with conventional stirred tanks. As a consequence, cell damage would be reduced as cell viability is highly sensitive to hydrodynamic conditions. To check these assumptions, an experimental setup was developed to measure the most important flow parameters (liquid surface level, liquid velocity, and liquid and wall shear stress) in two cellbag sizes (2 and 20 L) of Wave Bioreactors®. The measurements confirm in particular low shear stress values in both cellbags, indicating favorable hydrodynamic conditions for cell cultivation. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Haemodynamic assessment of human coronary arteries is affected by degree of freedom of artery movement

Abnormal haemodynamic parameters are associated with atheroma plaque progression and instability in coronary arteries. Flow recirculation, shear stress and pressure gradient are understood to be important pathogenic mediators in coronary disease. The effect of freedom of coronary artery movement on these parameters is still unknown. Fluid–structure interaction (FSI) simulations were carried out in 25 coronary artery models derived from authentic human coronaries in order to investigate the effect of degree of freedom of movement of the coronary arteries on flow recirculation, wall shear stress (WSS) and wall pressure gradient (WPG). Each FSI model had distinctive supports placed upon it. The quantitative and qualitative differences in flow recirculation, maximum wall shear stress (MWSS), areas of low wall shear stress (ALWSS) and maximum wall pressure gradient (MWPG) for each model were determined. The results showed that greater freedom of movement was associated with lower MWSS, smaller ALWSS, smaller flow recirculation zones and lower MWPG. With increasing percentage diameter stenosis (%DS), the effect of degree of freedom on flow recirculation and WSS diminished. Freedom of movement is an important variable to be considered for computational modelling of human coronary arteries, especially in the setting of mild to moderate stenosis.

Abbreviations: 3D: Three-dimensional; 3DR: Three-dimensional Reconstruction; 3D-QCA: Three-dimensional quantitative coronary angiography; ALWSS: Areas of low wall shear stress; CAD: Coronary artery disease; CFD: Computational fluid dynamics; %DS: Diameter stenosis percentage; EPCS: End point of counter-rotating streamlines; FSI: Fluid–structure interaction; IVUS: Intravascular ultrasound; LAD: Left anterior descending; MWSS: Maximum wall shear stress; SST: Shear stress transport; TAWSS: Time-averaged wall shear stress; WSS: wall shear stress; WPG: Wall pressure gradient; MWPG: Maximum wall pressure gradient; FFR: Fractional flow reserve; iFR: Instantaneous wave-free ratio     

Flow in the well: computational fluid dynamics is essential in flow chamber construction

A perfusion system was developed to generate well defined flow conditions within a well of a standard multidish. Human vein endothelial cells were cultured under flow conditions and cell response was analyzed by microscopy. Endothelial cells became elongated and spindle shaped. As demonstrated by computational fluid dynamics (CFD), cells were cultured under well defined but time varying shear stress conditions. A damper system was introduced which reduced pulsatile flow when using volumetric pumps. The flow and the wall shear stress distribution were analyzed by CFD for the steady and unsteady flow field. Usage of the volumetric pump caused variations of the wall shear stresses despite the controlled fluid environment and introduction of a damper system. Therefore the use of CFD analysis and experimental validation is critical in developing flow chambers and studying cell response to shear stress. The system presented gives an effortless flow chamber setup within a 6-well standard multidish.     

Effects of bubble–liquid two‐phase turbulent hydrodynamics on cell damage in sparged bioreactor

According to recent experimental studies on sparged bioreactors, significant cell damage may occur at the gas inlet region near the sparger. Although shear stress was proposed to be one of the potential causes for cell damage, detailed hydrodynamic studies at the gas inlet region of gas–liquid bioreactors have not been performed to date. In this work, a second‐order moment (SOM) bubble–liquid two‐phase turbulent model based on the two‐fluid continuum approach is used to investigate the gas–liquid hydrodynamics in the bubble column reactor and their potential impacts on cell viability, especially at the gas inlet region. By establishing fluctuation velocity and bubble–liquid two‐phase fluctuation velocities correlation transport equations, the anisotropy of two‐phase stresses and the bubble–liquid interactions are fully considered. Simulation results from the SOM model indicate that shear and normal stresses, turbulent energy dissipation rate, and the turbulent kinetic energy are generally smaller at the gas inlet region when compared with those in the fully developed region. In comparison, a newly proposed correlation expression, stress‐induced turbulent energy production (STEP), is found to correlate well with the unusually high cell death rate at the gas inlet region. Therefore, STEP, which represents turbulent energy transfer to a controlled volume induced by a combination of shear and normal stresses, has the potential to provide better explanation for increased cell death at the sparger region. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:48–58, 2014     

Rotating cylindrical filters used in perfusion cultures: CFD simulations and experiments

The particle and fluid dynamics in a rotating cylindrical filtration (RCF) system used for animal cell retention in perfusion processes was studied. A validated CFD model was used and the results gave numerical evidence of phenomena that had been earlier claimed, but not proven for this kind of application under turbulent and high mesh permeability conditions, such as bidirectional radial exchange flow (EF) through the filter mesh and particle (cells) lateral migration. Taylor vortices were shown to cause EF 10‐100 times higher than perfusion flow, indicating that EF is the main drag source, at least in early stages of RCF operation. Particle lateral migration caused a cell concentration reduction (CCR) near the filter surface of approximately 10%, contributing significantly to cell separation in RCF systems and giving evidence that the mesh sieving effect is not the sole phenomenon underlying cell retention in RCF systems. Filter rotation rate was shown to significantly affect both EF and CCR. A higher separation efficiency (measured experimentally at 2,000‐L bioreactor scale) and an enhanced CCR (predicted by the numerical simulations) were found for the same rotation rate range, indicating that there is an optimal operational space with practical consequences on RCF performance. Experimental data of a large‐scale perfusion run employing the simulated RCF showed high cell viabilities for over 100 days, which is probably related to the fact that the computed shear stress level in the system was shown to be relatively low (below 20 Pa under all tested conditions). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1093–1102, 2014     

Control of the shape of a thrombus-neointima-like structure by blood shear stress

Fluid mechanical factors are thought to influence vascular morphogenesis. Here we show how blood shear stress regulates the shape of a thrombus-neointima-like tissue on a polymer micro-cylinder implanted in the center of the rat vena cava with the micro-cylinder perpendicular to blood flow. In this model, the micro-cylinder is exposed to a laminarflow with a known shear stress field in the leading region and a vortexflow in the trailing region. At 1, 5, 10, 20, and 30 days after implantation, it was found that the micro-cylinder was encapsulated by a thrombus-neointima-like tissue with a streamlined body profile. The highest growth rate of the thrombus-neointima-like tissue was found along the trailing and leading stagnation edges of the micro-cylinder. Blood shear stress in the laminar flow region was inversely correlated with the rate of thrombus formation and cell proliferation, and the percentage of smooth muscle a actin-positive cells. These biological changes were also found in the trailing vortex flow region, which was associated with lowered shear stress. These results suggest that blood shear stress regulates the rate of thrombus and neointimal formation and, thus, influences the shape of the thrombus-neointima-like structure in the present model.     

Computational fluid dynamics simulation improves the design and characterization of a plug-flow-type scale-down reactor for microbial cultivation processes

The scale-up of bioprocesses remains one of the major obstacles in the biotechnology industry. Scale-down bioreactors have been identified as valuable tools to investigate the heterogeneities observed in large-scale tanks at the laboratory scale. Additionally, computational fluid dynamics (CFD) simulations can be used to gain information about fluid flow in tanks used for production. Here, we present the rational design and comprehensive characterization of a scale-down setup, in which a flexible and modular plug-flow reactor was connected to a stirred-tank bioreactor. With the help of CFD using the realizable k-ε model, the mixing time difference between a 20 and 4000 L bioreactor was evaluated and used as scale-down criterion. CFD simulations using a shear stress transport (SST) k-ω turbulence model were used to characterize the plug-flow reactor in more detail, and the model was verified using experiments. Additionally, the model was used to simulate conditions where experiments technically could not be performed due to sensor limitations. Nevertheless, verification is difficult in this case as well. This was the first time a scale-down setup was tested on high-cell-density Escherichia coli cultivations to produce industrially relevant antigen-binding fragments (Fab). Biomass yield was reduced by 11% and specific product yield was reduced by 20% during the scale-down cultivations. Additionally, the intracellular Fab fraction was increased by using the setup. The flexibility of the introduced scale-down setup in combination with CFD simulations makes it a valuable tool for investigating scale effects at the laboratory scale. More information about the large scale is still necessary to further refine the setup and to speed up bioprocess scale-up in the future.     

Influence of flow rate variation on the development of Escherichia coli biofilms

This work investigates the effect of flow rate variation on mass transfer and on the development of Escherichia coli biofilms on a flow cell reactor under turbulent flow conditions. Computational fluid dynamics (CFD) was used to assess the applicability of this reactor for the simulation of industrial and biomedical biofilms and the numerical results were validated by streak photography. Two flow rates of 374 and 242 L h^?1 (corresponding to Reynolds numbers of 6,720 and 4,350) were tested and wall shear stresses between 0.183 and 0.511 Pa were predicted in the flow cell reactor. External mass transfer coefficients of 1.38 × 10^?5 and 9.64 × 10^?6 m s^?1 were obtained for the higher and lower flow rates, respectively. Biofilm formation was favored at the lowest flow rate because shear stress effects were more important than mass transfer limitations. This flow cell reactor generates wall shear stresses that are similar to those found in some industrial and biomedical settings, thus it is likely that the results obtained on this work can be used in the development of biofilm control strategies in both scenarios.     

Microwell engineering characterization for mammalian cell culture process development

Experimentation in shaken microplate formats offers a potential platform technology for the rapid evaluation and optimization of cell culture conditions. Provided that cell growth and antibody production kinetics are comparable to those found in currently used shake flask systems then the microwell approach offers the possibility to obtain early process design data more cost effectively and with reduced material requirements. This work describes a detailed engineering characterization of liquid mixing and gas–liquid mass transfer in microwell systems and their impact on suspension cell cultures. For growth of murine hybridoma cells producing IgG1, 24‐well plates have been characterized in terms of energy dissipation (P/V) (via Computational Fluid Dynamics, CFD), fluid flow, mixing and oxygen transfer rate as a function of shaking frequency and liquid fill volume. Predicted k_La values varied between 1.3 and 29 h^?1; liquid‐phase mixing time, quantified using iodine decolorization experiments, varied from 1.7 s to 3.5 h; while the predicted P/V ranged from 5 to 35 W m^?3. CFD simulations of the shear rate predicted hydrodynamic forces will not be detrimental to cells. For hybridoma cultures however, high shaking speeds (>250 rpm) were shown to have a negative impact on cell growth, while a combination of low shaking speed and high well fill volume (120 rpm, 2,000 µL) resulted in oxygen limited conditions. Based on these findings a first engineering comparison of cell culture kinetics in microwell and shake flask formats was made at matched average energy dissipation rates. Cell growth kinetics and antibody titer were found to be similar in 24‐well microtiter plates and 250 mL shake flasks. Overall this work has demonstrated that cell culture performed in shaken microwell plates can provide data that is both reproducible and comparable to currently used shake flask systems while offering at least a 30‐fold decrease in scale of operation and material requirements. Linked with automation this provides a route towards the high throughput evaluation of robust cell lines under realistic suspension culture conditions. Biotechnol. Bioeng. 2010; 105: 260–275. © 2009 Wiley Periodicals, Inc.     

Dependence of leukocyte capture on instantaneous pulsatile flow

Atherosclerosis, an artery disease, is currently the leading cause of death in the United States in both men and women. The first step in the development of atherosclerosis involves leukocyte adhesion to the arterial endothelium. It is broadly accepted that blood flow, more specifically wall shear stress (WSS), plays an important role in leukocyte capture and subsequent development of an atherosclerotic plaque. What is less known is how instantaneous WSS, which can vary by up to 5 Pa over one cardiac cycle, influences leukocyte capture. In this paper we use direct numerical simulations (DNS), performed using an in-house code, to illustrate that leukocyte capture is different whether as a function of instantaneous or time-averaged blood flow. Specifically, a stenotic plaque is modeled using a computational fluid dynamics (CFD) solver through fully three-dimensional Navier-Stokes equations and the immersed boundary method. Pulsatile triphasic inflow is used to simulate the cardiac cycle. The CFD is coupled with an agent-based leukocyte capture model to assess the impact of instantaneous hemodynamics on stenosis growth. The computed wall shear stress agrees well with the results obtained with a commercial software, as well as with theoretical results in the healthy region of the artery. The analysis emphasizes the importance of the instantaneous flow conditions in evaluating the leukocyte rate of capture. That is, the capture rate computed from mean flow field is generally underpredicted compared to the actual rate of capture. Thus, in order to obtain a reliable estimate, the flow unsteadiness during a cardiac cycle should be taken into account.     

Cells in 3D matrices under interstitial flow: Effects of extracellular matrix alignment on cell shear stress and drag forces

Interstitial flow is an important regulator of various cell behaviors both in vitro and in vivo, yet the forces that fluid flow imposes on cells embedded in a 3D extracellular matrix (ECM), and the effects of matrix architecture on those forces, are not well understood. Here, we demonstrate how fiber alignment can affect the shear and pressure forces on the cell and ECM. Using computational fluid dynamics simulations, we show that while the solutions of the Brinkman equation accurately estimate the average fluid shear stress and the drag forces on a cell within a 3D fibrous medium, the distribution of shear stress on the cellular surface as well as the peak shear stresses remain intimately related to the pericellular fiber architecture and cannot be estimated using bulk-averaged properties. We demonstrate that perpendicular fiber alignment of the ECM yields lower shear stress and pressure forces on the cells and higher stresses on the ECM, leading to decreased permeability, while parallel fiber alignment leads to higher stresses on cells and increased permeability, as compared to a cubic lattice arrangement. The Spielman–Goren permeability relationships for fibrous media agreed well with CFD simulations of flow with explicitly considered fibers. These results suggest that the experimentally observed active remodeling of ECM fibers by fibroblasts under interstitial flow to a perpendicular alignment could serve to decrease the shear and drag forces on the cell.     

Investigation of air-liquid interfacial damage of animal cells in a falling film-flow device

A falling film-flow device was employed to expose animal cells to well-defined rapidly moving air-liquid interfaces. Cells (insect Sf-21) in the film flow suffered from severe mechanical damage with rapid cell death and lysis. The cell death rate was closely correlated with the momentum of the falling film but not the interfacial area, indicative of cell damage due to shear stress created by the falling film running into the bulk liquid, instead of direct interaction of the cells with the air-liquid interface.     

Computational models for wall shear stress estimation in scaffolds: a comparative study of two complete geometries

Fluid mechanical stimuli are known to upregulate cell differentiation and matrix formation. Since wall shear stress plays an important role various studies tried to estimate the scaffold fluid dynamic environment. However, because of the geometrical complexity, nearly all studies created their CFD model based on a submodel of the entire scaffold assuming that the model covers heterogeneity sufficiently. However to the authors' knowledge no study exist providing guidelines in this matter. In a previous study we demonstrated that submodels are influenced by the boundary conditions, inevitable when flow channels are chopped off. For the current study we therefore developed μCT based models of two complete scaffold geometries (one titanium and one hydroxyapatite). Imposing a 0.04 ml/min flow rate resulted in a surface area averaged wall shear stress of 1.41 mPa for titanium and 1.09 mPa for hydroxyapatite. In order to get insight in required model size we subdivided the domain in regions of different size. From our results we propose a model size between 6 and 10 times the average pore size. The wall shears stress should be calculated on a region at least one pore size away from the boundaries. These guidelines could be of use for computationally more costly simulations where it is not possible to simulate the complete scaffold domain.     

Aerodynamic study of time-trial helmets in cycling racing using CFD analysis

The aerodynamic drag of three different time-trial cycling helmets was analyzed numerically for two different cyclist head positions. Computational Fluid Dynamics (CFD) methods were used to investigate the detailed airflow patterns around the cyclist for a constant velocity of 15 m/s without wind. The CFD simulations have focused on the aerodynamic drag effects in terms of wall shear stress maps and pressure coefficient distributions on the cyclist/helmet system. For a given head position, the helmet shape, by itself, obtained a weak effect on a cyclist’s aerodynamic performance (<1.5%). However, by varying head position, a cyclist significantly influences aerodynamic performance; the maximum difference between both positions being about 6.4%. CFD results have also shown that both helmet shape and head position significantly influence drag forces, pressure and wall shear stress distributions on the whole cyclist’s body due to the change in the near-wake behavior and in location of corresponding separation and attachment areas around the cyclist.     

Effect of shear stress on migration and integrin expression in macaque trophoblast cells

During fetal development, trophoblast cells enter endometrial capillaries, migrate within the uterine vasculature, and eventually reside within spiral arteries of the uterus. This invasive activity is accompanied by upregulation of trophoblast beta1 integrin expression. Fluid mechanical shear stress regulates migration and expression of adhesion molecules in vascular endothelial cells, but nothing is known about the effects of shear stress on trophoblast cells. We tested the hypothesis that shear stress regulates the motility and beta1 integrin expression of trophoblast cells. Early gestation macaque trophoblast cells were cultured in 1 x 1-mm square cross-section capillary tubes within which the flow field was determined using three-dimensional computational fluid dynamic simulations. Trophoblast cells in the capillary tubes were exposed to a steady shear stress of 7.5, 15, or 30 dyn/cm2 for up to 24 h. In the absence of flow, trophoblast cells were highly dynamic with constant nondirectional positional shifts but with no net cell migration. Exposure of the cells to shear stress within 24-72 h of cell plating significantly increased the level of this activity and led to net cell migration in the direction of flow. Shear stress also increased the expression and altered the topography of beta1 integrin. These results suggest that shear stress regulates trophoblast motility and beta1 integrin expression in vitro.     

Macro-scale topology optimization for controlling internal shear stress in a porous scaffold bioreactor

Shear stress is an important physical factor that regulates proliferation, migration, and morphogenesis. In particular, the homeostasis of blood vessels is dependent on shear stress. To mimic this process ex vivo, efforts have been made to seed scaffolds with vascular and other cell types in the presence of growth factors and under pulsatile flow conditions. However, the resulting bioreactors lack information on shear stress and flow distributions within the scaffold. Consequently, it is difficult to interpret the effects of shear stress on cell function. Such knowledge would enable researchers to improve upon cell culture protocols. Recent work has focused on optimizing the microstructural parameters of the scaffold to fine tune the shear stress. In this study, we have adopted a different approach whereby flows are redirected throughout the bioreactor along channels patterned in the porous scaffold to yield shear stress distributions that are optimized for uniformity centered on a target value. A topology optimization algorithm coupled to computational fluid dynamics simulations was devised to this end. The channel topology in the porous scaffold was varied using a combination of genetic algorithm and fuzzy logic. The method is validated by experiments using magnetic resonance imaging readouts of the flow field.     

MECHANISMS OF FLUID SHEAR‐INDUCED INHIBITION OF POPULATION GROWTH IN A RED‐TIDE DINOFLAGELLATE1

Net population growth of some dinoflagellates is inhibited by fluid shear at shear stresses comparable with those generated during oceanic turbulence. Decreased net growth may occur through lowered cell division, increased mortality, or both. The dominant mechanism under various flow conditions was determined for the red‐tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge. Cell division and mortality were determined by direct observation of isolated cells in 0.5‐mL cultures that were shaken to generate unquantified fluid shear. Larger volume cultures were exposed to quantified laminar shear in Couette‐flow chambers (0.004–0.019 N·m ^{? 2} shear stress) and to unquantified flow in shaken flasks. In these larger cultures, cell division frequency was calculated from flow cytometric measurements of DNA·cell^?1. The mechanism by which shear inhibits net growth of L. polyedrum depends on shear stress level and growth conditions. Observations on the isolated cells showed that shaking inhibited growth by lowering cell division without increased mortality. Similar results were found for early exponential‐phase cultures exposed to the lowest experimental shear stress in Couette‐flow chambers. However, mortality occurred when a late exponential‐phase culture was exposed to the same low shear stress and was inferred to occur in cultures exposed to higher shear stresses. Elevated mortality in those treatments was confirmed using behavioral, morphological, and physiological assays. The results predict that cell division in L. polyedrum populations will be inhibited by levels of oceanic turbulence common for near‐surface waters. Shear‐induced mortality is not expected unless shear‐stress levels are unusually high or when cellular condition resembles late exponential/stationary phase cultures.     

Analysis of a high‐throughput cone‐and‐plate apparatus for the application of defined spatiotemporal flow to cultured cells

The shear stresses derived from blood flow regulate many aspects of vascular and immunobiology. In vitro studies on the shear stress‐mediated mechanobiology of endothelial cells have been carried out using systems analogous to the cone‐and‐plate viscometer in which a rotating, low‐angle cone applies fluid shear stress to cells grown on an underlying, flat culture surface. We recently developed a device that could perform high‐throughput studies on shear‐mediated mechanobiology through the rotation of cone‐tipped shafts in a standard 96‐well culture plate. Here, we present a model of the three‐dimensional flow within the culture wells with a rotating, cone‐tipped shaft. Using this model we examined the effects of modifying the design parameters of the system to allow the device to create a variety of flow profiles. We first examined the case of steady‐state flow with the shaft rotating at constant angular velocity. By varying the angular velocity and distance of the cone from the underlying plate we were able to create flow profiles with controlled shear stress gradients in the radial direction within the plate. These findings indicate that both linear and non‐linear spatial distributions in shear stress can be created across the bottom of the culture plate. In the transition and “parallel shaft” regions of the system, the angular velocities needed to provide high levels of physiological shear stress (5 Pa) created intermediate Reynolds number Taylor‐Couette flow. In some cases, this led to the development of a flow regime in which stable helical vortices were created within the well. We also examined the system under oscillatory and pulsatile motion of the shaft and demonstrated minimal time lag between the rotation of the cone and the shear stress on the cell culture surface. Biotechnol. Bioeng. 2013; 110: 1782–1793. © 2013 Wiley Periodicals, Inc.     

Computational fluid dynamics analysis of mixing and gas–liquid mass transfer in wave bag bioreactor

Single use bioreactors provide an attractive alternative to traditional deep-tank stainless steel bioreactors in process development and more recently manufacturing process. Wave bag bioreactors, in particular, have shown potential applications for cultivation of shear sensitive human and animal cells. However, the lack of knowledge about the complex fluid flow environment prevailing in wave bag bioreactors has so far hampered the development of a scientific rationale for their scale up. In this study, we use computational fluid dynamics (CFD) to investigate the details of the flow field in a 20-L wave bag bioreactor as a function of rocking angle and rocking speed. The results are presented in terms of local and mean velocities, mixing, and energy dissipation rates, which are used to create a process engineering framework for the scale-up of wave bag bioreactors. Proof-of-concept analysis of mixing and fluid flow in the 20-L wave bag bioreactor demonstrates the applicability of the CFD methodology and the temporal and spatial energy dissipation rates integrated and averaged over the liquid volume in the bag provide the means to correlate experimental volumetric oxygen transfer rates (k_La) data with power per unit volume. This correlation could be used as a rule of thumb for scaling up and down the wave bag bioreactors.     

Original article submission: Platelet stress accumulation analysis to predict thrombogenicity of an artificial kidney

An implantable artificial kidney using a hemofilter constructed from an array of silicon membranes to provide ultrafiltration requires a suitable blood flow path to ensure stable operation in vivo. Two types of flow paths distributing blood to the array of membranes were evaluated: parallel and serpentine. Computational fluid dynamics (CFD) simulations were used to guide the development of the blood flow paths. Pressure data from animal tests were used to obtain pulsatile flow conditions imposed in the transient simulations. A key consideration for stable operation in vivo is limiting platelet stress accumulation to avoid platelet activation and thrombus formation. Platelet stress exposure was evaluated by CFD particle tracking methods through the devices to provide distributions of platelet stress accumulation. The distributions of stress accumulation over the duration of a platelet lifetime for each device revealed that stress accumulation for the serpentine flow path exceeded levels expected to cause platelet activation while the accumulated stress for the parallel flow path was below expected activation levels.