In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology

Abstract

In hematologic diseases, such as sickle cell disease (SCD) and hemolytic uremic syndrome (HUS), pathological biophysical interactions among blood cells, endothelial cells, and soluble factors lead to microvascular occlusion and thrombosis. Here, we report an in vitro "endothelialized" microfluidic microvasculature model that recapitulates and integrates this ensemble of pathophysiological processes. Under controlled flow conditions, the model enabled quantitative investigation of how biophysical alterations in hematologic disease collectively lead to microvascular occlusion and thrombosis. Using blood samples from patients with SCD, we investigated how the drug hydroxyurea quantitatively affects microvascular obstruction in SCD, an unresolved issue pivotal to understanding its clinical efficacy in such patients. In addition, we demonstrated that our microsystem can function as an in vitro model of HUS and showed that shear stress influences microvascular thrombosis/obstruction and the efficacy of the drug eptifibatide, which decreases platelet aggregation, in the context of HUS. These experiments establish the versatility and clinical relevance of our microvasculature-on-a-chip model as a biophysical assay of hematologic pathophysiology as well as a drug discovery platform.

Dynamic interactions ofPlasmodiumspp. with vascular endothelium

“…150 Microfluidic models have also allowed for analysis of cellular interactions within microvascular networks (10-100mm) using either de novo generated vascular networks in 3D matrices 151 or in patterned devices devoid of matrices. 152 The microfluidic models would be ideal for studying endothelial-IRBC interactions in capillaries (5-15mm) and post-capillary venules (30-70mm) where hostpathogen interactions occur in vivo. The hemodynamics of IRBC-endothelial interaction in microfluidic microvessels are likely to vary significantly from the more traditional 2D cultures, including changes in shear stress through high variability in hematocrit, viscosity and parasitemia within microvessels which can lead to vessel obstruction, changes in mechanoreceptor signaling and in turn endothelial function.…”

A standalone perfusion platform for drug testing and target validation in micro-vessel networks

“…The endothelial coating within the rectangular channels gradually detached from the channel walls at the four sharp corners, possibly due to increasing intercellular tension and decreasing cell adhesion to PDMS surface. The presence of areas with high tension at the corners of rectangular channels, which are often neglected in other configurations, 8 may result in non-physiological cell responses. In contrast, endothelial coating within circular channels remained stably attached.…”

“…Current microfluidic approaches either culture endothelial cells simply on the bottom of a micro-channel [5][6][7] or coat endothelial cells around a rectangular channel with sharp corners. [6][7][8][9][10] These approaches can establish a vasculature quickly in defined conditions but ignore the artifacts generated by the sharp corners on the endothelialized lumen. Although remodeling of collagen gels allows for formation of circular lumens, fragility of these gels limits the complexity of branching structures and levels of branching, enabling creation of structures that branch only to the first order, 6 in contrast to the native vasculature that follows fractal rules in branching.…”

Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in sickle cell disease

“…[2][3][4] However, the relevance of these mechanisms is not completely understood in humans. As non-invasive in vivo imaging in humans is limited by low-resolution, [5][6][7] there is a need for in vitro approaches 8 that can allow visualization of single cell events in flowing human blood. Here, we introduce quantitative microfluidic fluorescence microscopy (qMFM) that enables visualization of cellular interactions in human blood flowing through silicone microfluidic channels.…”

“…However, these approaches were limited by the use of isolated SS-RBCs 10 or the inability to visualize cellular interactions at single cell resolution 11 and distinguish different cell types that constitute the vaso-occlusive plug. 8 We introduce qMFM (Figure 1), which enables visualization of molecular interactions between neutrophils and platelets at single cell resolution in SS blood. The methods used are described in detail in the Online Supplementary Information.…”