Dynamics of blood flow and thrombus formation in a multi-bypass microfluidic ladder network

Abstract

The reaction dynamics of a complex mixture of cells and proteins, such as blood, in branched circulatory networks within the human microvasculature or extravascular therapeutic devices such as extracorporeal oxygenation machine (ECMO) remains ill-defined. In this report we utilize a multi-bypass microfluidics ladder network design with dimensions mimicking venules to study patterns of blood platelet aggregation and fibrin formation under complex shear. Complex blood fluid dynamics within multi-bypass networks under flow were modeled using COMSOL. Red blood cells and platelets were assumed to be non-interacting spherical particles transported by the bulk fluid flow, and convection of the activated coagulation factor II, thrombin, was assumed to be governed by mass transfer. This model served as the basis for predicting formation of local shear rate gradients, stagnation points and recirculation zones as dictated by the bypass geometry. Based on the insights from these models, we were able to predict the patterns of blood clot formation at specific locations in the device. Our experimental data was then used to adjust the model to account for the dynamical presence of thrombus formation in the biorheology of blood flow. The model predictions were then compared to results from experiments using recalcified whole human blood. Microfluidic devices were coated with the extracellular matrix protein, fibrillar collagen, and the initiator of the extrinsic pathway of coagulation, tissue factor. Blood was perfused through the devices at a flow rate of 2 µL/min, translating to physiologically relevant initial shear rates of 300 and 700 s^-1 for main channels and bypasses, respectively. Using fluorescent and light microscopy, we observed distinct flow and thrombus formation patterns near channel intersections at bypass points, within recirculation zones and at stagnation points. Findings from this proof-of-principle ladder network model suggest a specific correlation between microvascular geometry and thrombus formation dynamics under shear. This model holds potential for use as an integrative approach to identify regions susceptible to intravascular thrombus formation within the microvasculature as well as extravascular devices such as ECMO.

Keywords: mass transfer; microfluidic network; multi-bypass ladder; platelets; shear; thrombin.

Figure 1

Modeling human blood flow and thrombus formation dynamics within a multi-bypass ladder network. Parameters of a multi-bypass microfluidic ladder network device design (a) and an experimental prototype as visualized by differential interference contrast (DIC) microscopy (b). Network features two main channels and ten bypasses (bp 1–10); micrograph shows the first four bypasses. Black bars indicate a stoppered inlet and outlet; red arrows depict the direction of blood flow. Stoppered inlets and outlets were re-opened during the washing step.

Figure 2

Prediction of thrombus formation within the ladder network: nucleation and evolution. Computer simulation of blood flow velocity profile (a; µL/min), shear rate profile (b; s⁻¹) and the initial blood cell distribution profile (c). A combination of velocity streamlines and shear rate gradient predicted a higher probability for the nucleation site for thrombus formation on the channel walls immediately downstream of intersections between main channels and bypasses (highlighted with black circles) (d). Computer simulations repeated in the presence of an obstruction to predict adjusted blood cell distribution profile during clot growth and thrombin convection mol/m³ (e) and combination of thrombin convection profile (purple) over bulk flow velocity streamlines and shear rate profile (f). Shear rates below 30 s⁻¹ were subtracted from the shear rate heat maps on the overlays (d and f) for visibility of velocity streamlines.

Figure 3

Temporal thrombus growth within ladder network. DiOC₆-labeled whole human blood was perfused at a 2 µL/min flow rate through a PDMS-ladder network coated with collagen and tissue factor; real-time images of thrombus formation were recorded using differential interference contrast, DIC (a) and fluorescence microscopy (b). Networks were subsequently washed with modified Hepes-Tyrodes buffer for 20 min prior to DIC imaging (c). Representative images shown, n = 5. Total surface areas of thrombi per bypass were quantified and normalized to bp1 (d).

Figure 4

Prediction of the effect of thrombus formation within a ladder network. Computer simulations of the effects of thrombus formation on the dynamics of blood flow dynamics were performed at discrete time and bypass-to-bypass locations (a). The changes in shear profile (b; s⁻¹), platelet distribution profile (c) and concentration profile of thrombin (d; nmol/s) at each bypass were calculated as a function of time. Arrows indicate regions of measurement integration.

Figure 5

Prediction of the effect of the flow recirculation on platelet aggregation and fibrin formation. Computer simulation of the streamlines of the bulk blood flow through the network ladder predicts flow distortions at the intersection of the first bypass (bp 1; resulting in a zone of recirculation), at the stoppered channel (black bar), and at channel 2 (a). Simulation of blood cell transport during thrombus formation: <50% of bypass obstruction, left, and >50% bypass obstruction, right (b); shear rate profile (c) and thrombin convection profile mol/m³ (d) within these regions.

Figure 6

Effect of flow recirculation on platelet aggregation and fibrin formation. Dynamics of whole human blood flow at the intersection where the first bypass meets a stoppered channel, creating a recirculation zone (black bar); platelet aggregation was recorded using differential interference contrast, DIC and fluorescence microscopy in real time (a) while fibrin was imaged following a washing step (b). Representative images shown, n = 3.

Figure 7

An integrated approach to study thrombus formation within a microfluidic network. Integrated approach of creating simulations of blood flow velocity, shear and blood cell distribution profiles to predict the site of thrombus formation. Modeling was repeated to account for the dynamical response of blood rheology and thrombin generation as a function of temporal thrombus formation.