Less Deformable Erythrocyte Subpopulations Biomechanically Induce Endothelial Inflammation in Sickle Cell Disease.

Sickle cell disease (SCD) is canonically characterized by reduced red blood cell (RBC) deformability leading to microvascular obstruction and inflammation. While the biophysical properties of sickle RBCs are known to influence SCD vasculopathy, the contribution of poor RBC deformability to endothelial dysfunction has yet to be fully explored. Leveraging interrelated in vitro and in silico approaches, we introduce a new paradigm of SCD vasculopathy in which poorly deformable sickle RBCs directly cause endothelial dysfunction via mechanotransduction, where endothelial cells sense and pathophysiologically respond to aberrant physical forces independently of microvascular obstruction, adhesion, or hemolysis. We demonstrate that perfusion of sickle RBCs or pharmacologically-dehydrated healthy RBCs into small venule-sized "endothelialized" microfluidics leads to pathologic physical interactions with endothelial cells that directly induce inflammatory pathways. Using a combination of computational simulations and large venule-sized endothelialized microfluidics, we observed that perfusion of heterogeneous sickle RBC subpopulations of varying deformability, as well as suspensions of dehydrated normal RBCs admixed with normal RBCs leads to aberrant margination of the less-deformable RBC subpopulations towards the vessel walls, causing localized, increased shear stress. Increased wall stress is dependent on the degree of subpopulation heterogeneity and oxygen tension and leads to inflammatory endothelial gene expression via mechanotransductive pathways. Our multifaceted approach demonstrates that the presence of sickle RBCs with reduced deformability leads directly to pathological physical (i.e., direct collisions and/or compressive forces) and shear-mediated interactions with endothelial cells and induces an inflammatory response, thereby elucidating the ubiquity of vascular dysfunction in SCD.

Stiffness based enrichment of leukemia cells using microfluidics.

To improve the survival rate of cancer patients, new diagnosis strategies are necessary to detect lower levels of cancer cells before and after treatment regimens. The scarcity of diseased cells, particularly in residual disease after treatment, demands highly sensitive detection approaches or the ability to enrich the diseased cells in relation to normal cells. We report a label-free microfluidic approach to enrich leukemia cells from healthy cells using inherent differences in cell biophysical properties. The microfluidic device consists of a channel with an array of diagonal ridges that recurrently compress and translate flowing cells in proportion to cell stiffness. Using devices optimized for acute T cell leukemia model Jurkat, the stiffer white blood cells were translated orthogonally to the channel length, while softer leukemia cells followed hydrodynamic flow. The device enriched Jurkat leukemia cells from white blood cells with an enrichment factor of over 760. The sensitivity, specificity, and accuracy of the device were found to be > 0.8 . The values of sensitivity and specificity could be adjusted by selecting one or multiple outlets for analysis. We demonstrate that low levels of Jurkat leukemia cells (1 in 10 4 white blood cells) could be more quickly detected using flow cytometry by using the stiffness sorting pre-enrichment. In a second mode of operation, the device was implemented to sort resistive leukemia cells from both drug-sensitive leukemia cells and normal white blood cells. Therefore, microfluidic biomechanical sorting can be a useful tool to enrich leukemia cells that may improve downstream analyses.