Cell-free Layer Research Articles

Plasma expander viscosity effects on red cell-free layer thickness after moderate hemodilution

The objective of the study was to investigate the effects of plasma viscosity after hemodilution on the thickness of the erythrocyte cell free layer (CFL) and on the interface between the flowing column of erythrocytes and the vascular endothelium. The erythrocyte CFL thickness was measured in the rat cremaster muscle preparation. Plasma viscosity was modified in an isovolemic hemodilution, in which the systemic hematocrit (Hctsys) was lowered to 30%. The plasma expanders (PE) of similar nature and different viscosities were generated by glutaraldehyde polymerization of human serum albumin (HSA) at various molar ratios glutaraldehyde to HSA: (i) unpolymerized HSA; (ii) PolyHSA24:1, molar ratio = 24 and (iii) PolyHSA60:1, molar ratio = 60. The HSA viscosities determined at 200 s(-1) were 1.1, 4.2 and 6.0 dyn x cm(-2), respectively. CFL thickness, vessel diameter and blood flow velocity were measured, while volumetric flow, shear rate and stress were calculated. Hemodilution with PolyHSA60:1 increased plasma viscosity and the blood showed marked shear thinning behavior. CFL thickness decreased as plasma viscosity increased after hemodilution; thus the CFL thickness with HSA and PolyHSA24:1 increased compared to baseline. Conversely, the CFL thickness of PolyHSA60:1 was not different from baseline. Blood flow increased with both PolyHSA's compared to baseline. Wall shear rate and shear stress increased for PolyHSA60:1 compared to HSA and PolyHSA24:1, respectively. In conclusion, PE viscosity determined plasma viscosity after hemodilution and affected erythrocyte column hydrodynamics, changing the velocity profile, CFL thickness, and wall shear stress. This study relates the perfusion caused by PolyHSA60:1 to hemodynamic changes induced by the rheological properties of blood diluted with PolyHSA60:1.

Effect of plasma expander viscosity on the cell free layer

The effect of low and high viscosity hemodilution with plasma expanders on the extent of the cell free layer (CFL) width was analyzed in the microcirculation of the exteriorized cremaster muscle preparation of Sprague-Dawley male rats. Anesthetized animals were subjected to 40% hemodilution by blood volume, using 5% human serum albumin (HSA) or 6% Hetastarch (hydroxyethyl starch 670 kDa). Arterioles (n=5 for each treatment) were investigated. Mean arterial pressure, heart rate, vessel flow velocity and CFL width were measured at baseline and 5, 20 and 40 min post-exchange transfusion. Blood and plasma viscosity was determined from terminal blood collections. CFL width and pseudoshear rate, diameter and flow, normalized to baseline, were significantly elevated at all post-exchange assessments. Peripheral vascular resistance decreased. The increase of the CFL width was greater with HSA by comparison with Hetastarch hemodilution (p<0.05). Hetastarch blood and plasma viscosities increased significantly compared to those of HSA (p<0.05). This study shows that CFL widths are influenced by plasma expander viscosity, a phenomenon proportional to the increase in molecular weight of the colloids in solution.

Mathematical Model of Blood Flow in Small Blood Vessel in the Presence of Magnetic Field

A mathematical model for blood flow in the small blood vessel in the presence of magnetic field is presented in this paper. We have modeled the two phase model for the blood flow consists of a central core of suspended erythrocytes and cell-free layer surrounding the core. The system of differential equations has been solved analytically. We have obtained the result for velocity, flow rate and effective viscosity in presence of peripheral layer and magnetic field .All the result has been obtained and discussed through graphs.

Modulation of NO Bioavailability by Temporal Variation of the Cell-Free Layer Width in Small Arterioles

The cell-free layer exhibits dynamic characteristics in the time domain that may be capable of altering nitric oxide (NO) bioavailability in small arterioles. However, this effect has not been fully elucidated. This study utilized a computational model on NO transport to predict how temporal variations in the layer width could modulate NO bioavailability in the arterioles. Data on the layer width was acquired from high-speed video recordings in arterioles (ID=48.4±1.8μm) of the rat cremaster muscle. We found that when wall shear stress response was not considered, the layer variability could lead to a slight decrease (1.6-6.6%) in NO bioavailability that was independent of transient changes in NO scavenging rate. Conversely, the transient response in wall shear stress and NO production rate played a dominant role in reversing this decline such that a significant augmentation (5.3-21.0%) in NO bioavailability was found with increasing layer variability from 24.6 to 63.8%. This study highlighted the importance of the temporal changes in wall shear stress and NO production rate caused by the layer width variations in prediction of NO bioavailability in arterioles.

Blood Flow and Cell-Free Layer in Microvessels

Blood is modeled as a suspension of red blood cells using the dissipative particle dynamics method. The red blood cell membrane is coarse-grained for efficient simulations of multiple cells, yet accurately describes its viscoelastic properties. Blood flow in microtubes ranging from 10 to 40 μm in diameter is simulated in three dimensions for values of hematocrit in the range of 0.15-0.45 and carefully compared with available experimental data. Velocity profiles for different hematocrit values show an increase in bluntness with an increase in hematocrit. Red blood cell center-of-mass distributions demonstrate cell migration away from the wall to the tube center. This results in the formation of a cell-free layer next to the tube wall corresponding to the experimentally observed Fahraeus and Fahraeus-Lindqvist effects. The predicted cell-free layer widths are in agreement with those found in in vitro experiments; the results are also in qualitative agreement with in vivo experiments. However, additional features have to be taken into account for simulating microvascular flow, e.g., the endothelial glycocalyx. The developed model is able to capture blood flow properties and provides a computational framework at the mesoscopic level for obtaining realistic predictions of blood flow in microcirculation under normal and pathological conditions.

Asymmetry of blood flow and cancer cell adhesion in a microchannel with symmetric bifurcation and confluence

Bifurcations and confluences are very common geometries in biomedical microdevices. Blood flow at microchannel bifurcations has different characteristics from that at confluences because of the multiphase properties of blood. Using a confocal micro-PIV system, we investigated the behaviour of red blood cells (RBCs) and cancer cells in microchannels with geometrically symmetric bifurcations and confluences. The behaviour of RBCs and cancer cells was strongly asymmetric at bifurcations and confluences whilst the trajectories of tracer particles in pure water were almost symmetric. The cell-free layer disappeared on the inner wall of the bifurcation but increased in size on the inner wall of the confluence. Cancer cells frequently adhered to the inner wall of the bifurcation but rarely to other locations. Because the wall surface coating and the wall shear stress were almost symmetric for the bifurcation and the confluence, the result indicates that not only chemical mediation and wall shear stress but also microscale haemodynamics play important roles in the adhesion of cancer cells to the microchannel walls. These results provide the fundamental basis for a better understanding of blood flow and cell adhesion in biomedical microdevices.

A comparative study of histogram-based thresholding methods for the determination of cell-free layer width in small blood vessels

We have recently proposed a computer-based method utilizing a thresholding algorithm (the Otsu method) to provide a convenient way of measuring the cell-free layer width in vivo and in vitro. However, this method does not seem to be a universal method that can be applied to all microvascular studies. Thus, we examined four different histogram-based thresholding algorithms (Otsu, intermode, minimum and second peak) to provide a technical suggestion on the selection of a suitable thresholding algorithm for the cell-free layer measurement. All the measurements were taken in microvascular flows in the rat cremaster muscle recorded with a high-speed camera. The width of the cell-free layer manually measured was compared with that determined by the automated method utilizing the four thresholding algorithms. With our experimental system, results showed that the cell-free layer width determined by the minimum algorithm was in best accordance with the manual measurement. We concluded that the accuracy of the automated methods for determination of the cell-free layer width would depend on the image quality, in particular on the contrast between the red blood cell core and background, which might differ due to the different microscopic setup. Therefore, one may need to examine several appropriate thresholding methods when selecting the best suitable algorithm for the experimental conditions.

Effect of Cell-Free Layer Variation on Arteriolar Wall Shear Stress

Relationship between a cell-free layer and wall shear stress (WSS) in small arterioles has been of interest in microcirculatory research. However, influence of temporal variation in the cell-free layer width on the WSS in vivo has not been fully elucidated. In this study, we tested the hypothesis that the layer variation would increase the WSS, and this effect would be enhanced by red blood cell aggregation. The cell-free layer width in arterioles (29.5–67.1 μm ID) in rat cremaster muscles were obtained with a high-speed video camera, and the layer width data were introduced into WSS estimation. Dextran 500 was administrated to elevate the aggregation level of red blood cells to those seen in normal human blood. The variation of the layer was quantified by the variability (coefficient of variation), and its effect on WSS was studied under normal and reduced flow conditions. We found that the dextran-induced red blood cell aggregation significantly elevated the variability (p < 0.01) at low pseudoshear rates of 9.2 ± 0.6 s−1. The WSS estimated without taking account of the variability showed underestimation of its value than that of with consideration of the variability under all flow conditions, and this effect became more pronounced with increasing the variability. The variation of the cell-free layer should, therefore, be considered in the determination of the WSS particularly in the presence of red blood cell aggregation under reduced flow condition.

Effect of systemic hematocrit on volume flow and cell free layer width in arterioles

The effect of hematocrit on effective viscosity is well known for in vitro systems but the effect in vivo is less clear due to the formation of a cell free layer (CFL) at the vascular wall that may influence vascular tone. The CFL and volumetric flow rate (Q) were assessed in rat cremaster arterioles (20–40 μm) during systemic hemodilution and hemoconcentration. Systemic hematocrit (Hctsys) was increased stepwise by 5% from 40 to 75% during hemoconcentration and decreased by 5% from 40 to 20% during hemodilution. The CFL was measured with a high speed video camera and custom built software. Velocity and vessel diameter measurements were obtained from the video files using a dual‐window cross correlation technique and calipers. The CFL values were 5.4± 0.2, 3.1±0.1 and 0.9±0.1 μm at 20, 40 and 75% Hctsys, respectively. The Q values were 14.5, 9.0±0.6 and 4.9±0.4 nl/sec at 20, 40 and 75% Hctsys, respectively. Overall, Q decreased 66% from 20 to 75% Hctsys. The vessel diameter did not change significantly as Hctsys was reduced from 40% to 20% and from 75 to 40%. The arterial pressure was 17% higher at 40% and 32% higher at 75% as compared to 20% Hctsys. The flow change was inversely proportionate to Hctsys change when corrected for differences in arterial pressure. Apparently the change in CFL width did not induce vascular responses in these vessels due to simultaneous changes in other factors.

Modulation of NO bioavailability by temporal variability of cell‐free layer width in the arterioles

The formation of a cell (erythrocyte)‐free layer near the vessel wall during blood flow is capable of attenuating NO consumption by the red blood cells and thus improves the preservation of NO in the smooth muscle for eliciting vasodilatory responses. This study utilized a transient NO transport mathematical model to predict how temporal variability exhibited by the layer width and its relevant transient physiological responses could modulate NO preservation by this diffusion barrier at high pseudoshear rates (291.5 ± 13.5 s−1). Data on cell‐free layer width was experimentally acquired from arterioles (48.4 ± 2.3 μm) of the rat cremaster muscle. We found that a time‐varying diffusion barrier could lead to a slight decrease (2% – 6%) in NO bioavailability and this effect was independent of transient changes in NO scavenging rate in the blood lumen. Conversely, transient changes in wall shear stress and accompanying NO release rate played a dominant role in reversing this decline such that an augmentation in NO bioavailability of between 3% – 15% was found. However this increase was not sufficient to induce any significant change in vascular tone. This study highlighted the importance of accounting for transient wall shear stress effect on NO release rate as mediated by the layer width temporal variability when assessing the physiological implication of the layer on NO bioavailability in arterioles.

Effect of erythrocyte aggregation and flow rate on cell-free layer formation in arterioles

Formation of a cell-free layer is an important dynamic feature of microcirculatory blood flow, which can be influenced by rheological parameters, such as red blood cell aggregation and flow rate. In this study, we investigate the effect of these two rheological parameters on cell-free layer characteristics in the arterioles (20-60 mum inner diameter). For the first time, we provide here the detailed temporal information of the arteriolar cell-free layer in various rheological conditions to better describe the characteristics of the layer variation. The rat cremaster muscle was used to visualize arteriolar flows, and the extent of aggregation was raised by dextran 500 infusion to levels seen in normal human blood. Our results show that cell-free layer formation in the arterioles is enhanced by a combination of flow reduction and red blood cell aggregation. A positive relation (P < 0.005) was found between mean cell-free layer widths and their corresponding SDs for all conditions. An analysis of the frequency and magnitudes of cell-free layer variation from their mean value revealed that the layer deviated with significantly larger magnitudes into the red blood cell core after flow reduction and dextran infusion (P < 0.05). In accordance, the disparity of cell-free layer width distribution found in opposite radial directions from its mean became greater with aggregation in reduced flow conditions. This study shows that the cell-free layer width in arterioles is dependent on both flow rate and red blood cell aggregability, and that the temporal variations in width are asymmetric with a greater excursion into the red blood cell core than toward the vessel wall.

Fast and continuous plasma extraction from whole human blood based on expanding cell-free layer devices

This paper presents promising microfluidic devices designed for continuous and passive extraction of plasma from whole human blood. These designs are based on red cells lateral migration and the resulting cell-free layer locally expanded by geometric singularities such as an enlargement of the channel or a cavity adjacent to the channel. After an explanation of flow patterns, different tests are described that confirm the advantages of both proposed singularities, providing a 1.5 and 2X increase in extraction yield compared to a reference device, for 1:20 diluted blood at 100 microL/min. Devices have also been successively optimized, with extraction yields up to 17.8%, and biologically validated for plasma extraction, with no protein loss or denaturation, no hemolysis and with excellent cell purity. Finally, the dilution effect has been experimentally investigated.

Technique for Real-Time Measurements of Endothelial Permeability in a Microfluidic Membrane Chip Using Laser-Induced Fluorescence Detection

Characterizing permeability of the endothelium that lines blood vessels and heart valves provides fundamental physiological information and is required to evaluate uptake of drugs and other biomolecules. However, current techniques used to measure permeability, such as Transwell insert assays, do not account for the recognized effects of fluid flow-induced shear stress on endothelial permeability or are inherently low-throughput. Here we report a novel on-chip technique in a two-layer membrane-based microfluidic platform to measure real-time permeability of endothelial cell monolayers on porous membranes. Bovine serum albumin (a model protein) conjugated with fluorescein isothiocyanate was delivered to an upper microchannel by pressure-driven flow and was forced to permeate a poly(ethylene terephthalate) membrane into a lower microchannel, where it was detected by laser-induced fluorescence. The concentration of the permeate at the point of detection varied with channel flow rates in agreement to less than 1% with theoretical analyses using a pore flow model. On the basis of the model, a sequential flow rate stepping scheme was developed and applied to obtain the permeability of cell-free and cell-bound membrane layers. This technique is a highly sensitive, novel microfluidic approach for measuring endothelial permeability in vitro, and the use of micrometer-sized channels offers the potential for parallelization and increased throughput compared to conventional shear-based permeability measurement methods.

Mathematical modelling of the cell-depleted peripheral layer in the steady flow of blood in a tube

In an earlier paper, Moyers-Gonzalez et al. [J. Fluid. Mech. 617 (2008), 327-354] used kinetic theory to derive a non-homogeneous haemorheological model and applied this to simulate the properties of steady flow of blood in a tube. By adjusting the tube haematocrit to match that of the experimental fitted curve of Pries et al. [Circ. Res. 67 (1990), 826-834] the authors showed that it was possible to quantitatively predict the apparent viscosity values presented in a later paper by Pries et al. [Am. J. Physiol. 263 (1992), 1770-1778]. In the present paper, it is the discharge haematocrit rather than the tube haematocrit that is prescribed. We further develop the predictive capacities of the original model of Moyers-Gonzalez et al. [J. Fluid. Mech. 617 (2008), 327-354] by introducing a cell-free peripheral layer next to the tube wall where, following the ideas of Sharan and Popel [Biorheology 38 (2001), 415-428], dissipation in this layer is accounted for by allowing the viscosity there to exceed that of plasma. Using both the apparent viscosity data of Pries et al. [Am. J. Physiol. 263 (1992), 1770-1778] and the relative tube haematocrit relation proposed by Sharan and Popel [Biorheology 38 (2001), 415-428], we predict the thickness of the cell-free layer and the relative viscosity in this layer. The predicted thickness of the cell-free layer as a function of both a pseudo-shear rate and the tube diameter for 45% haematocrit blood is shown to be in very close conformity with the experimental measurements of Reinke et al. [Am. J. Physiol. 253 (1987), 540-547]. With increasing discharge haematocrit the cell-free layer thickness is shown to decrease, as observed in several experimental papers [Bugliarello and Hayden, Trans. Soc. Rheol. VII (1963), 209-230, Bugliarello and Sevilla, Biorheology 7 (1970), 85-107, Soutani et al., Am. J. Physiol. 268 (1995), 1959-1965]. Our prediction of the relative viscosity in the cell-free layer shows a similar trend to that computed by Sharan and Popel [Biorheology 38 (2001), 415-428]. Finally, for sufficiently large pseudo-shear rates it is shown that the Deborah number (a non-dimensional relaxation time) may be taken to be a constant, thus greatly simplifying our haemorheological model and allowing for a partially analytic solution to the problem of steady non-homogeneous flow of blood in a tube.

A low-dimensional model for the red blood cell

The red blood cell (RBC) is an important determinant of the rheological properties of blood because of its predominant number density, special mechanical properties and dynamics. Here, we develop a new low-dimensional RBC model based on dissipative particle dynamics (DPD). The model is constructed as a closed-torus-like ring of 10 colloidal particles connected by wormlike chain springs combined with bending resistance. Each colloidal particle is represented by a single DPD particle with a repulsive core. The model is able to capture the essential mechanical properties of RBCs, and allows for economical exploration of the rheology of RBC suspensions. Specifically, we find that the linear and non-linear elastic deformations of healthy and malaria-infected cells match those obtained in optical tweezers experiments. Through simulations of some key features of blood flow in vessels, i.e., the cell-free layer (CFL), the Fahraeus effect and the Fahraeus-Lindqvist effect, we verify that the new model captures the essential shear flow properties of real blood, except for capillaries of sizes comparable to the cell diameter. Finally, we investigate the influence of a geometrical constriction in the flow on the enhancement of the downstream CFL. Our results are in agreement with recent experiments.

High flow rate microfluidic device for blood plasma separation using a range of temperatures

A hybrid microfluidic device that uses hydrodynamic forces to separate human plasma from blood cells has been designed and fabricated and the advantageous effects of temperature and flow rates are investigated in this paper. The blood separating device includes an inlet which is reduced by approximately 20 times to a small constrictor channel, which then opens out to a larger output channel with a small lateral channel for the collection of plasma. When tested the device separated plasma from whole blood using a wide range of flow rates, between 50 microl min(-1) and 200 microl min(-1), at the higher flow rates injected by hand and at temperatures ranging from 23 degrees C to 50 degrees C, the latter resulting in an increase in the cell-free layer of up to 250%. It was also tested continuously using between 5% and 40% erythrocytes in plasma and whole blood without blocking the channels or hemolysis of the cells. The mean percentage of plasma collected after separation was 3.47% from a sample of 1 ml. The percentage of cells removed from the plasma varied depending on the flow rate used, but at 37 degrees C ranged between 95.4 +/- 1% and 97.05 +/- 05% at 100 microl min(-1) and 200 microl min(-1), respectively. The change in temperature also had an effect on the number of cells removed from the plasma which was between 93.5 +/- 0.65% and 97.01 +/- 0.3% at 26.9 degrees C and 37 degrees C, respectively, using a flow rate of 100 microl min(-1). Due to its ability to operate in a wide range of conditions, it is envisaged that this device can be used in in vitro 'lab on a chip' applications, as well as a hand-held point of care (POC) device.

Microfluidic hydrogel layers with multiple gradients to stimulate and perfuse three-dimensional neuronal cell cultures

We present a simple and easy to handle PDMS microfluidic device for neuronal cell culture studies in three-dimensional hydrogel scaffolds. The hydrogel is structured in parallel layers to reconstruct cell layers close to the natural environment. Dissociated cortical neurons of embryonic rats have been cultured in 0.5% w/v agarose including 0.2% w/v alginate. The cells formed neurite networks through neighboring cell free hydrogel layers. The cell culture showed neurite outgrowth in the microfluidic channel over more than seven days in vitro without perfusion. Culturing neurons in hydrogel layers surrounded by a liquid phase containing culture medium resulted in denser neuronal networks.

Passive microfluidic devices for plasma extraction from whole human blood

Our goal is to analyze and compare different continuous microfluidic principles dedicated to plasma extraction from hardly diluted human blood for lab-on-chip applications. First, the strengths and weaknesses of various emerging passive microfluidic methods (microfiltration- and centrifugation-based methods) were analyzed. Various devices were designed, microfabricated and tested with beads or blood. Filtration may be efficient, but with a high sample dilution, low flow rate and optimized geometry. Due to fast cell clogging, this remains a short-term solution. Separation effects resulting from centrifugal acceleration in curved channel flows are hindered by Dean vortices and anyhow are not pronounced with blood. An innovative device is then proposed and investigated experimentally. This is based on the lateral migration of red cells and the resulting cell-free layer, which is used to supply geometric singularities (an ear-cavity or a corner-edge) and locally enhance the clear plasma region. A maximum extraction of 10.7% is obtained for 1/20 diluted blood, injected at 100 μL/min in the corner-edge design.

Three-dimensional computational modeling of multiple deformable cells flowing in microvessels

Three-dimensional (3D) computational modeling and simulation are presented on the motion of a large number of deformable cells in microchannels. The methodology is based on an immersed boundary method, and the cells are modeled as liquid-filled elastic capsules. The model retains two important features of the blood flow in the microcirculation, that is, the particulate nature of blood and deformation of the erythrocytes. The tank-treading and tumbling motion and the lateral migration, as observed for erythrocytes in dilute suspension, are briefly discussed. We then present results on the motion of multiple cells in semidense suspension and study how their collective dynamics leads to various physiologically relevant processes such as the development of the cell-free layer and the Fahraeus-Lindqvist effect. We analyze the 3D trajectory and velocity fluctuations of individual cell in the suspension and the plug-flow velocity profile as functions of the cell deformability, hematocrit, and vessel size. The numerical results allow us to directly obtain various microrheological data, such as the width of the cell-free layer, and the variation in the apparent blood viscosity and hematocrit over the vessel cross section. We then use these results to calculate the core and plasma-layer viscosity and show that the two-phase (or core-annular) model of blood flow in microvessels underpredicts the blood velocity obtained in the simulations by as much as 40%. Based on a posteriori analysis of the simulation data, we develop a three-layer model of blood flow by taking into consideration the smooth variation in viscosity and hematocrit across the interface of the cell-free layer and the core. We then show that the blood velocity predicted by the three-layer model agrees very well with that obtained from the simulations.

Effect of erythrocyte aggregation and flow rate on temporal variation of cell‐free layer width in arterioles

The cell‐free layer (CFL) is a dynamic feature of microcirculatory blood flow and its variability is commonly described in terms of standard deviation (SD). However, CFL width in an arteriole follows a non‐Gaussian distribution imposed by asymmetrical nature of its physical environment, which implies that more detailed statistical analyses are required to better interpret CFL characteristics. In this study, rat cremaster muscle was used to visualize arteriolar flows and erythrocyte aggregability was raised by dextran infusion to levels seen in normal human blood. The effects of aggregation and flow reduction on CFL characteristics in arterioles (ID 20‐50 μm) were investigated. A positive relation was found between mean CFL widths and their corresponding SD and was predominantly enhanced by flow reduction. Both normalized mean and SD of CFL width increased significantly at low pseudoshear rates (17.1 ± 6.1 s−1), especially in the presence of aggregation. The variability of the CFL width was further analyzed in terms of its frequency and magnitude of deviations from its mean value, extending either towards the vessel wall or into the erythrocyte core. There was no significant effect of either flow condition or aggregation on the frequency of CFL deviations, whereas the magnitude of deviations significantly increased after flow reduction and dextran infusion (P &lt; 0.05), especially into the erythrocyte core.