Coronary Flow Rate Adds Predictive Capability for FFR Assessment.

A non-invasive risk assessment tool capable of stratifying coronary artery stenosis into high and low risk would reduce the number of patients who undergo invasive FFR, the current gold standard procedure for assessing coronary artery disease. Current statistic-based models that predict if FFR is above or below the threshold for physiological significance rely completely on anatomical parameters, such as percent diameter stenosis (%DS), resulting in models not accurate enough for clinical application. The inclusion of coronary artery flow rate (CFR) was added to an anatomical-only logistic regression model to quantify added predictive value. Initial hypothesis testing on a cohort of 96 coronary artery segments with some degree of stenosis found higher mean CFR in a group with low FFR < 0.8 (µ = 2.37 ml/s) compared to a group with high FFR > 0.8 (µ = 1.85 ml/s) (p-value = 0.046). Logistic regression modeling using both %DS and CFR (AUC = 0.78) outperformed logistic regression models using either only %DS (AUC = 0.71) or only CFR (AUC = 0.62). Including physiological parameters in addition to anatomical parameters are necessary to improve statistical based models for assessing high or low FFR.

Acoustofluidic-mediated molecular delivery to human T cells with a three-dimensional-printed flow chamber.

Cell-based therapies have garnered significant interest to treat cancer and other diseases. Acoustofluidic technologies are in development to improve cell therapy manufacturing by facilitating rapid molecular delivery across the plasma membrane via ultrasound and microbubbles (MBs). In this study, a three-dimensional (3D) printed acoustofluidic device was used to deliver a fluorescent molecule, calcein, to human T cells. Intracellular delivery of calcein was assessed after varying parameters such as MB face charge, MB concentration, flow channel geometry, ultrasound pressure, and delivery time point after ultrasound treatment. MBs with a cationic surface charge caused statistically significant increases in calcein delivery during acoustofluidic treatment compared to MBs with a neutral surface charge (p < 0.001). Calcein delivery was significantly higher with a concentric spiral channel geometry compared to a rectilinear channel geometry (p < 0.001). Additionally, calcein delivery was significantly enhanced at increased ultrasound pressures of 5.1 MPa compared to lower ultrasound pressures between 0-3.8 MPa (p < 0.001). These results demonstrate that a 3D-printed acoustofluidic device can significantly enhance intracellular delivery of biomolecules to T cells, which may be a viable approach to advance cell-based therapies.

Blood residence time to assess significance of coronary artery stenosis.

Coronary artery stenosis is a narrowing of coronary lumen space caused by an atherosclerotic lesion. Fractional flow reserve (FFR) is the gold standard metric to assess physiological significance of coronary stenosis, but requires an invasive procedure. Computational modeling in conjunction with patient-specific imaging demonstrates formation of regions of recirculatory flow distal to a stenosis, increasing mean blood residence time relative to uninhibited flow. A new computational parameter, mean blood residence time (BloodRT), was computed for 100 coronary artery segments for which FFR was known. A threshold for BloodRT was determined to assess the physiological significance of a stenosis, analogous to diagnostic threshold for FFR. Model sensitivity and specificity of BloodRT for diagnosis of hemodynamically significant coronary stenosis was 98% and 96% respectively, compared with FFR. When applied to clinical practice, this could potentially allow practicing cardiologists to accurately assess the severity of coronary stenosis without resorting to invasive techniques.

Impact of Bi-Axial Shear on Atherogenic Gene Expression by Endothelial Cells.

This study demonstrated the effects of the directionality of oscillatory wall shear stress (WSS) on proliferation and proatherogenic gene expression (I-CAM, E-Selectin, and IL-6) in the presence of inflammatory mediators leukotriene B4 (LTB4) and bacterial lipopolysaccharide (LPS) from endothelial cells grown in an orbiting culture dish. Computational fluid dynamics (CFD) was applied to quantify the flow in the dish, while an analytical solution representing an extension of Stokes second problem was used for validation. Results indicated that WSS magnitude was relatively constant near the center of the dish and oscillated significantly (0-0.9 Pa) near the side walls. Experiments showed that LTB4 dominated the shear effects on cell proliferation and area. Addition of LPS didn't change proliferation, but significantly affected cell area. The expression of I-CAM1, E-Selectin and IL-6 were altered by directional oscillatory shear index (DOSI, a measure of the biaxiality of oscillatory shear), but not shear magnitude. The significance of DOSI was further reinforced by the strength of its interactions with other atherogenic factors. Hence, directionality of shear appears to be an important factor in regulating gene expression and provides a potential explanation of the propensity for increased vascular lesions in regions in the arteries with oscillating biaxial flow.

An orbital shear platform for real-time, in vitro endothelium characterization.

Electrical impedance techniques have been used to characterize endothelium morphology, permeability, and motility in vitro. However, these impedance platforms have been limited to either static endothelium studies and/or induced laminar fluid flow at a constant, single shear stress value. In this work, we present a microfabricated impedance sensor for real-time, in vitro characterization of human umbilical vein endothelial cells (HUVECs) undergoing oscillatory hydrodynamic shear. Oscillatory shear was applied with an orbital shaker and the electrical impedance was measured by a microfabricated impedance chip with discrete electrodes positioned at radial locations of 0, 2.5, 5.0, 7.5, 10.0, and 12.5 mm from the center of the chip. Depending on their radial position within the circular orbital platform, HUVECs were exposed to shear values ranging between 0.6 and 6.71 dyne/cm(2) (according to numerical simulations) for 22 h. Impedance spectra were fit to an equivalent circuit model and the trans-endothelial resistance and monolayer's capacitance were extracted. Results demonstrated that, compared to measurements acquired before the onset of shear, cells at the center of the platform that experienced low steady shear stress (∼2.2 dyne/cm(2) ) had an average change in trans-endothelial resistance of 6.99 ± 4.06% and 1.78 ± 2.40% change in cell capacitance after 22 hours of shear exposure; cells near the periphery of the well (r = 12.5 mm) experienced transient shears (2.5-6.7 dyne/cm(2) ) and exhibited a greater change in trans-endothelial resistance (24.2 ± 10.8%) and cell capacitance (4.57 ± 5.39%). This study, demonstrates that the orbital shear platform provides a simple system that can capture and quantify the real-time cellular morphology as a result of induced shear stress. The orbital shear platform presented in this work, compared to traditional laminar platforms, subjects cells to more physiologically relevant oscillatory shear as well as exposes the sample to several shear values simultaneously. Biotechnol. Bioeng. 2016;113: 1336-1344. © 2015 Wiley Periodicals, Inc.

Factors affecting cellulose hydrolysis based on inactivation of adsorbed enzymes.

The rate of enzymatic hydrolysis of cellulose reaction is known to decrease significantly as the reaction proceeds. Factors such as reaction temperature, time, and surface area of substrate that affect cellulose conversion were analyzed relative to their role in a mechanistic model based on first order inactivation of adsorbed cellulases. The activation energies for the hydrolytic step and inactivation step were very close in magnitude: 16.3 kcal mol(-1) for hydrolysis and 18.0 kcal mol(-1) for inactivation, respectively. Therefore, increasing reaction temperature would cause a significant increase in the inactivation rate in addition to the catalytic reaction rate. Vmax,app was only 20% or less of the value at 72 h compared to at 2h as a result of inactivation of adsorbed cellulases, suggesting prolonged hydrolysis is not an efficient way to improve cellulose hydrolysis. Hydrolysis rate increased with corresponding increases in available substrate surface binding area.

Relative extents of activity loss between enzyme-substrate interactions and combined environmental mechanisms.

Enzymatic hydrolysis of biomass undergoes a significant decrease in rate, which is often attributed to activity loss of enzyme during the incubation. Activity loss due to both interaction with substrate (for example inactivation of adsorbed enzyme) and all combined environmental mechanisms in a substrate free buffer solution were compared in this study. Enzyme-substrate interactions contributed more towards the overall activity loss than did the combined environmental sources as evidenced from three independent metrics. (1) Relative extents of inactivation were higher for enzyme-substrate interactions than for environmental mechanisms. (2) Apparent half-lives (1.37-11.01 h) following interaction with substrate were relatively small compared to environmental inactivation, which was 21.5h. (3) The inactivation rate constant for enzyme-substrate interactions (0.56 h(-1)) was 46 times higher than that of environmental inactivation (0.0123 h(-1)). These results suggest enzyme-substrate interaction is the main cause of cellulase activity loss and contributes significantly to the slow rate of hydrolysis.

Mixing analysis of PCS slurries in a horizontal scraped surface bioreactor.

Horizontal rotating reactors offer many advantages for enzymatic hydrolysis of viscous biomass slurries; however, they do not provide homogenous mixtures since motion is only in the angular direction. Multi-directional mixing is important for dispersing enzymes and carrying products away from reaction sites. The objective here was to experimentally quantify mixing times and axial dispersion coefficients in a horizontal rotating bioreactor. Mixing times were of the same order as reaction times, indicating that enzymatic hydrolysis could be as much controlled by diffusion and mixing effects as by the complex reaction mechanism. The dispersion coefficient for the highest solids slurry was 20× less than the lowest solids slurry, which is indicative of the difference in free water and the magnitude change of viscosity with relatively small addition of solids. The slow mixing times and low dispersion may be an acceptable tradeoff with significantly lower power requirements compared to a conventional vertical reactor.

Effects of biaxial oscillatory shear stress on endothelial cell proliferation and morphology.

Wall shear stress (WSS) on anchored cells affects their responses, including cell proliferation and morphology. In this study, the effects of the directionality of pulsatile WSS on endothelial cell proliferation and morphology were investigated for cells grown in a Petri dish orbiting on a shaker platform. Time and location dependent WSS was determined by computational fluid dynamics (CFD). At low orbital speed (50 rpm), WSS was shown to be uniform (0-1 dyne/cm(2)) across the bottom of the dish, while at higher orbital speed (100 and 150 rpm), WSS remained fairly uniform near the center and fluctuated significantly (0-9 dyne/cm(2)) near the side walls of the dish. Since WSS on the bottom of the dish is two-dimensional, a new directional oscillatory shear index (DOSI) was developed to quantify the directionality of oscillating shear. DOSI approached zero for biaxial oscillatory shear of equal magnitudes near the center and approached one for uniaxial pulsatile shear near the wall, where large tangential WSS dominated a much smaller radial component. Near the center (low DOSI), more, smaller and less elongated cells grew, whereas larger cells with greater elongation were observed in the more uniaxial oscillatory shear (high DOSI) near the periphery of the dish. Further, cells aligned with the direction of the largest component of shear but were randomly oriented in low magnitude biaxial shear. Statistical analyses of the individual and interacting effects of multiple factors (DOSI, shear magnitudes and orbital speeds) showed that DOSI significantly affected all the responses, indicating that directionality is an important determinant of cellular responses.

Deactivation of individual cellulase components.

Deactivation extents of cellobiohydrolase, endoglucanase, and a total cellulase mixture (Spezyme CP) were studied independently as functions of incubating time and mixing intensity. It was found that the decrease in total cellulase activity was more strongly related to deactivation of cellobiohydrolase 1 (CBH1) than endoglucanase. The mass-averaged shear in orbiting flasks at 50, 150, and 250rpm was quantified by computational fluid dynamics and was two-orders smaller than shear in typical stirred tanks. Endoglucanase activity did not change significantly with mixing speed, but CBH1 and total cellulase activities were 10-25% higher at 250rpm compared to the lower speeds after a 24-h incubation. Total deactivation due to mechanical mixing (∼20%) may be too low to account for all the rate reduction during cellulose hydrolysis. Thermal deactivation was independent of enzyme concentration while deactivation due to mechanical stress decreased when cellulase loading increased over 0.15 filterpaperunit/ml.

Kinetic modeling of cellulose hydrolysis with first order inactivation of adsorbed cellulase.

Enzymatic hydrolysis involves complex interaction between enzyme, substrate, and the reaction environment, and the complete mechanism is still unknown. Further, glucose release slows significantly as the reaction proceeds. A model based on Langmuir binding kinetics that incorporates inactivation of adsorbed cellulase was developed that predicts product formation within 10% of experimental results for two substrates. A key premise of the model, with experimental validation, suggests that V(max) decreases as a function of time due to loss of total available enzyme as adsorbed cellulases become inactivated. Rate constants for product formation and enzyme inactivation were comparable to values reported elsewhere. A value of k(2)/K(m) that is several orders of magnitude lower than the rate constant for the diffusion-controlled encounter of enzyme and substrate, along with similar parameter values between substrates, implies a common but undefined rate-limiting step associated with loss of enzyme activity likely exists in the pathway of cellulose hydrolysis.

Scaled-up separation of cellobiohydrolase1 from a cellulase mixture by ion-exchange chromatography.

Enzymatic hydrolysis of cellulose often involves cellulases produced by Trichoderma reesei, of which cellobiohydrolase1 (CBH1) is the most abundant (about 60% of total cellulases) and plays an important role in the hydrolysis of crystalline cellulose. A method for separating sufficient quantities from the bulk cellulase cocktail is highly desirable for many studies, such as those that aim to characterize binding and hydrolysis kinetics of CBH1. In this work, CBH1 was separated from other Spezyme CP cellulases by ion-exchange chromatography using an efficient modification of a smaller scale process. The ion-exchange column was connected to a vacuum manifold system to provide a steady flow through parallel columns and thus achieve scale-up for enzyme separation. With five 5-mL columns running in parallel, about 55 mg of CBH1 was separated from 145 mg of Spezyme CP in a single separation. Step elution was used to replace the continuous gradient used at smaller scale. The purified CBH1 was collected in the fraction eluted with a buffer containing 0.33 M salt and showed comparable purity and activity as the enzyme purified by a fast protein liquid chromatography system. The stability of separated CBH1 was studied for up to 2 days and good thermal stability was observed. Separated CBH1 also showed both high adsorption to bacterial microcrystalline cellulose with ~4 µmol/g maximum adsorption and a K(a) of 5.55 ± 2.34 µM(-1) , and good hydrolytic activity based on atomic force microscopy observations that show a reduction in fiber height.

Spatial and temporal resolution of shear in an orbiting petri dish.

It is well documented that physiological and morphological properties of anchored cells are influenced by fluid shear stress. Common orbital shakers provide a means of simultaneously applying shear stress to cells for tens to hundreds of cases by loading the shaker with multiple dishes. However, the complex flow in orbiting dishes is amenable to analytical solution for resolving shear created by the fluid motion only for simplified conditions. The only existing quantification of shear in this flow is an equation that estimates a constant scalar value of shear for the entire surface of the dish. In practice, wall shear stress (WSS) will be oscillatory rather than steady due to the travelling waveform and will vary across the surface of the dish at any instant in time. This article presents a computational model that provides complete spatial and temporal resolution of WSS over the bottom surface of a dish throughout the orbital cycle. The model is reasonably well validated by the analytical solution, with resultant WSS magnitudes that are within 0.99 ± 0.42 dyne/cm(2) . The model results were compared to tangential WSS magnitudes obtained using one-dimensional optical velocimetry at discreet locations on the bottom of an orbiting dish. The experimental minimum and maximum WSS at 1 mm from the center of the dish were 6 and 7 dyne/cm(2) , respectively, whereas WSS generated from the computational model ranged from 0.5 to 8.5 dyne/cm(2) . The experimental minimum and maximum WSS at 12 mm from the center of the dish were 6 and 16 dyne/cm(2) , respectively, whereas WSS generated from the computational model ranged from 0.5 to 14 dyne/cm(2) . Discrepancies between the experimental and computational data may be attributed to a sparse sampling rate for the experimental probe, a sharp gradient at the sample area which could cause the unidirectional probe to be inaccurate if its location were not precise, and too few particles to track and a scattering of the signal by the free surface when the liquid is shallow.

Role of shear stress in endothelial cell morphology and expression of cyclooxygenase isoforms.

OBJECTIVE: The goal of this study was to examine the effect of chronic heterogeneous shear stress, applied using an orbital shaker, on endothelial cell morphology and the expression of cyclooxygenases 1 and 2. METHODS AND RESULTS: Porcine aortic endothelial cells were plated on fibronectin-coated Transwell plates. Cells were cultured for up to 7 days either under static conditions or on an orbital shaker that generated a wave of medium inducing shear stress over the cells. Cells were fixed and stained for the endothelial surface marker CD31 or cyclooxygenases 1 and 2. En face confocal microscopy and scanning ion conductance microscopy were used to show that endothelial cells were randomly oriented at the center of the well, aligned with shear stress nearer the periphery, and expressed cyclooxygenase-1 under all conditions. Lipopolysaccharide induced cyclooxygenase-2 and the production of 6-keto-prostaglandin F(1α) in all cells. CONCLUSIONS: Cyclooxygenase-1 is expressed in endothelial cells cultured under chronic shear stress of high or low directionality.

Hydrodynamic modulation of embryonic stem cell differentiation by rotary orbital suspension culture.

Embryonic stem cells (ESCs) can differentiate into all somatic cell types, but the development of effective strategies to direct ESC fate is dependent upon defining environmental parameters capable of influencing cell phenotype. ESCs are commonly differentiated via cell aggregates referred to as embryoid bodies (EBs), but current culture methods, such as hanging drop and static suspension, yield relatively few or heterogeneous populations of EBs. Alternatively, rotary orbital suspension culture enhances EB formation efficiency, cell yield, and homogeneity without adversely affecting differentiation. Thus, the objective of this study was to systematically examine the effects of hydrodynamic conditions created by rotary orbital shaking on EB formation, structure, and differentiation. Mouse ESCs introduced to suspension culture at a range of rotary orbital speeds (20-60 rpm) exhibited variable EB formation sizes and yields due to differences in the kinetics of cell aggregation. Computational fluid dynamic analyses indicated that rotary orbital shaking generated relatively uniform and mild shear stresses (< or =2.5 dyn/cm(2)) within the regions EBs occupied in culture dishes, at each of the orbital speeds examined. The hydrodynamic conditions modulated EB structure, indicated by differences in the cellular organization and morphology of the spheroids. Compared to static culture, exposure to hydrodynamic conditions significantly altered the gene expression profile of EBs. Moreover, varying rotary orbital speeds differentially modulated the kinetic profile of gene expression and relative percentages of differentiated cell types. Overall, this study demonstrates that manipulation of hydrodynamic environments modulates ESC differentiation, thus providing a novel, scalable approach to integrate into the development of directed stem cell differentiation strategies.

Enzymatic saccharification and viscosity of sawdust slurries following ultrasonic particle size reduction.

Results in a previous study showed up to a 55% increase in saccharification rates when the initial particle size range decreased from 590 < x < 850 microm down to 33 < x < 75 microm. The smaller particle sizes also lowered the viscosity of the slurry 50-fold (for an equivalent initial solids concentration). In this study, ultrasonic irradiation was employed to further reduce the particle size of sawdust slurries below the ranges in the previous study in an attempt to further increase enzymatic saccharification rates and lower the slurry viscosity. Average particle sizes were reduced to less than 1 microm under the conditions tested. Surprisingly, the amount and rates of sugar released in this study with the approximately 1 microm particles is comparable (maximum glucose release of 30%) to, but no better than that seen for particle sizes in the range of 33 < or = x < or = 75 microm (maximum glucose release of 31%). Also surprisingly, the viscosity increased as the average particle sizes in the slurries decreased, which is opposite to the trend in the previous study. For example, there was an approximately threefold increase in the viscosity between unsonicated samples with a range of 10 < or = x < or = 75 microm and sonicated samples with a range of 0.05 < or = x < or = 12 microm. This is attributed to the variations in surface characteristics of the particles which were characterized here using X-ray diffraction profiles and SEM pictures.

Computationally determined shear on cells grown in orbiting culture dishes.

A new computational model, using computational fluid dynamics (CFD), is presented that describes fluid behavior in cylindrical cell culture dishes resulting from motion imparted by an orbital shaker apparatus. This model allows for the determination of wall shear stresses over the entire area of the bottom surface of a dish (representing the growth surface for cells in culture) which was previously too complex for accurate quantitative analysis. Two preliminary cases are presented that show the complete spatial resolution of the shear on the bottom of the dishes. The maximum shear stress determined from the model is compared to an existing simplified point function that provides only the maximum value. Furthermore, this new model incorporates seven parameters versus the four in the previous technique, providing improved accuracy. Optimization of computational parameters is also discussed.

A self-feeding roller bottle for continuous cell culture.

The concept of a self-feeding roller bottle that delivers a continuous supply of fresh media to cells in culture, which is mechanically simplistic and works with existing roller apparatuses, is presented here. A conventional roller bottle is partitioned into two chambers; one chamber contains the fresh culture media reservoir, and the other contains the cell culture chamber. A spiroid of tubing inside the fresh media reservoir acts as a pump when the bottle rotates on its horizontal axis, continuously delivering fresh media through an opening in the partition to the cell culture chamber. The modified bottle proved capable of maintaining steady-state cell densities of a hybridoma cell line over the 10-day period tested, although at lower densities than reached during batch operation due to the continuous volume dilution. Steady-state density proved to be controllable by adjusting the perfusion rate, which changes with the rotation rate of the bottle. Specific antibody production rate is as much as 3.7 times the rate in conventional roller bottles operating with intermittent batch feeding.

Reintroduced solids increase inhibitor levels in a pretreated corn stover hydrolysate.

Following detoxification of the liquid hydrolysate produced in a corn stover pretreatment process, inhibitor levels are seen to increase with the re-addition of solids for the ensuing hydrolysis and fermentation processes. The solids that were separated from the slurry before detoxification of the liquor contain approx 60% (w/w) moisture, and contamination occurs owing to the diffusion of inhibitors from the moisture entrained in the porous structure of the corn stover solids into the bulk fluid. This evidence suggests the need for additional separation and detoxification steps to purge residual inhibitors entrained in the moisture in the solids. An overliming process to remove furans from the hydrolysate failed to reduce total organic acids concentration, so acids were removed by treatment with an activated carbon powder. Smaller carbon doses proved more efficient in removing organic acids in terms of grams of acid removed per gram of carbon powder. Sugar adsorption by the activated carbon powder was minimal.