In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology
Michelle Tsai, Ashley Kita, Joseph Leach, Ross Rounsevell, James N. Huang, Joel Moake, Russell E. Ware, Daniel A. Fletcher, Wilbur A. Lam
Michelle Tsai, Ashley Kita, Joseph Leach, Ross Rounsevell, James N. Huang, Joel Moake, Russell E. Ware, Daniel A. Fletcher, Wilbur A. Lam
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Technical Advance Hematology

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.

Authors

Michelle Tsai, Ashley Kita, Joseph Leach, Ross Rounsevell, James N. Huang, Joel Moake, Russell E. Ware, Daniel A. Fletcher, Wilbur A. Lam

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Figure 3

Decreased flow and microchannel occlusion due to activation with the inflammatory cytokine TNF-α.

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Decreased flow and microchannel occlusion due to activation with the inf...
(A) Brightfield imaging and epifluorescence using R6G, a fluorescent dye that preferentially stains leukocytes and platelets, revealed that when whole blood is flowed into the microdevice cultured with HLMVECs at postcapillary venular flow conditions, flow is steady overall, with occasional rolling but few adherent leukocytes. (B) When endothelial cells were activated with TNF-α before whole blood was flowed into the system, brightfield and R6G staining revealed a slight decrease in overall flow velocity, with occasional microchannel obstructions as well as an increase in adherent leukocytes. (C) TNF-α activation of both the endothelial cells and whole blood revealed a dramatic increase in microchannel obstruction, with subsequent decrease in overall flow likely due to a combination of increased adhesion and cell stiffness. Scale bars: 30 μm. (D) The addition of 0.5 μm fluorescent beads mixed into the whole blood samples enabled velocity quantification in each microchannel. The centerline bead velocity was measured for each channel and averaged for each data point. Compared with the control condition (no TNF-α activation), the average centerline velocity per microchannel (n = 32 microchannels/device) was significantly decreased when endothelial cells were preactivated with TNF-α (P < 0.05) and even more so when both endothelial cells and whole blood were preincubated with TNF-α (P < 0.005). Error bars show SD. (E) The percentage of microchannels completely obstructed due to aggregates of leukocytes increased with TNF-α of endothelial cells and increased even further with TNF-α activation of both the endothelial cells and whole blood.