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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.

SUBMITTER: Tsai M

PROVIDER: S-EPMC3248292 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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

Tsai Michelle M Kita Ashley A Leach Joseph J Rounsevell Ross R Huang James N JN Moake Joel J Ware Russell E RE Fletcher Daniel A DA Lam Wilbur A WA

The Journal of clinical investigation 20111212 1

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 ...[more]

PMID: 22156199

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Project description:Abnormal red blood cell (RBC) deformability contributes to hemolysis, thrombophilia, inflammation, and microvascular occlusion in various circulatory diseases. A quantitative and objective assessment of microvascular occlusion mediated by RBCs with abnormal deformability would provide valuable insights into disease pathogenesis and therapeutic strategies. To that end, we present a new functional microfluidic assay, OcclusionChip, which mimics two key architectural features of the capillary bed in the circulatory system. First, the embedded micropillar arrays within the microchannel form gradient microcapillaries, from 20 μm down to 4 μm, which mimic microcapillary networks. These precisely engineered microcapillaries retain RBCs with impaired deformability, such that stiffer RBCs occlude the wider upstream microcapillaries, while less stiff RBCs occlude the finer downstream microcapillaries. Second, the micropillar arrays are coupled with two side passageways, which mimic the arteriovenous anastomoses that act as shunts in the capillary bed. These side microfluidic anastomoses prevent microchannel blockage, and enable versatility and testing of clinical blood samples at near-physiologic hematocrit levels. Further, we define a new generalizable parameter, Occlusion Index (OI), which is an indicative index of RBC deformability and the associated microcapillary occlusion. We demonstrate the promise of OcclusionChip in diverse pathophysiological scenarios that result in impaired RBC deformability, including mercury toxin, storage lesion, end-stage renal disease, malaria, and sickle cell disease (SCD). Hydroxyurea therapy improves RBC deformability and increases fetal hemoglobin (HbF%) in some, but not all, treated patients with SCD. HbF% greater than 8.6% has been shown to improve clinical outcomes in SCD. We show that OI associates with HbF% in 16 subjects with SCD. Subjects with higher HbF levels (HbF > 8.6%) displayed significantly lower OI (0.88% ± 0.10%, N = 6) compared with those with lower HbF levels (HbF ≤ 8.6%) who displayed greater OI (3.18% ± 0.34%, N = 10, p < 0.001). Moreover, hypoxic OcclusionChip assay revealed a significant correlation between hypoxic OI and subject-specific sickle hemoglobin (HbS) level in SCD. OcclusionChip enables versatile in vitro assessment of microvascular occlusion mediated by RBCs in a wide range of clinical conditions. OI may serve as a new parameter to evaluate the efficacy of treatments improving RBC deformability, including hemoglobin modifying drugs, anti-sickling agents, and genetic therapies.

Project description:Pathological processes in hematologic diseases originate at the single-cell level, often making measurements on individual cells more clinically relevant than population averages from bulk analysis. For this reason, flow cytometry has been an effective tool for single-cell analysis of properties using light scattering and fluorescence labeling. However, conventional flow cytometry cannot measure cell mechanical properties, alterations of which contribute to the pathophysiology of hematologic diseases such as sepsis, diabetic retinopathy, and sickle cell anemia. Here we present a high-throughput microfluidics-based 'biophysical' flow cytometry technique that measures single-cell transit times of blood cell populations passing through in vitro capillary networks. To demonstrate clinical relevance, we use this technique to characterize biophysical changes in two model disease states in which mechanical properties of cells are thought to lead to microvascular obstruction: (i) sepsis, a process in which inflammatory mediators in the bloodstream activate neutrophils and (ii) leukostasis, an often fatal and poorly understood complication of acute leukemia. Using patient samples, we show that cell transit time through and occlusion of microfluidic channels is increased for both disease states compared to control samples, and we find that mechanical heterogeneity of blood cell populations is a better predictor of microvascular obstruction than average properties. Inflammatory mediators involved in sepsis were observed to significantly affect the shape and magnitude of the neutrophil transit time population distribution. Altered properties of leukemia cell subpopulations, rather than of the population as a whole, were found to correlate with symptoms of leukostasis in patients-a new result that may be useful for guiding leukemia therapy. By treating cells with drugs that affect the cytoskeleton, we also demonstrate that their transit times could be significantly reduced. Biophysical flow cytometry offers a low-cost and high-throughput diagnostic and drug discovery platform for hematologic diseases that affect microcirculatory flow.

Dataset Information

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

Publications

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

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