Developing microfluidic systems to resolve longstanding hematological questions

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Ciciliano, Jordan C.
Lam, Wilbur A.
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Recent research has revealed that cells dynamically sense and respond to their physical microenvironments. In hematology, it has been shown that shear mediated red blood cell (RBC) deformation results in release, and that platelets attenuate contraction force based on substrate stiffness. Blood cells exist in a dynamic fluidic microenvironment, and they also interact with various matrices such as fibrin clots and the extracellular matrix proteins. The objective of this dissertation is thus to create microfluidic systems in which the biophysical and biochemical aspects of hematological processes are independently investigated toward the aim of discovering new solution spaces for diagnostics and therapeutics. To that end, this work presents novel microfluidic systems: 1) an “endothelial”-ized, T-junction fluidic to elucidate the biophysical processes that define the mechanism of action of the ferric chloride thrombosis model and 2) microfluidic devices with single-micron features (pillars and canals) to examine the effects of physical interactions between blood cell and geometrically relevant, non-biological matrices. Using the T-junction fluidic I resolved the mechanism of action of the most commonly used thrombosis injury model – the ferric chloride- thrombosis model. I show that the mechanism of action of ferric chloride is non-biological in nature. Rather, the chemical induces charge-based aggregation of blood cells that then triggers the conventional clotting cascade. This finding both reconciles discrepancies in the findings in the literature, and cautions researchers against using this injury model, especially for the study of clot initiation. My work also highlights the importance of considering mass transfer properties in biological processes. The second suite of devices recapitulates the physical dimensions of vascular matrices that blood cells interact with. By combining electron beam lithography, photolithography, and soft lithography, I considered hematological questions that were previously technologically infeasible. I found that the physical presence of a micropillar array creates a shear microgradient that leads to the localized adherence and aggregation of platelets that propagates to stem blood flow in the absence of exogenous agonists. Furthermore, I showed RBC fragmentation (schistocyte formation) real-time in an in vitro system by creating microcanals that forced RBCs to deform through a small cross-sectional area over various lengths of time. This further shows that RBC fragmentation is a time dependent process, contrary to what is historically cited in medical literature. Finally, by perfusing neutrophils through the microcanals, I show that mechanical forces can cause neutrophils to fragment into membranous debris that has neutrophil extracellular trap (NET)-like properties. This system provides insights into the synergy between hemolytic anemias, sepsis, and thrombosis. The mechanistic understandings gained by creating systems that successfully decouple the biophysical and biological aspects of blood cells, as is done in this work, can result in enhanced understanding of the etiology of pathologies, improved diagnostic assays for blood cell activity, and new targets for therapeutics.
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