Abnormal cell mechanical stiffness can point to the development of various diseases including cancers and infections. We report a new microfluidic technique for continuous cell separation utilizing variation in cell stiffness. We use a microfluidic channel decorated by periodic diagonal ridges that compress the flowing cells in rapid succession. The compression in combination with secondary flows in the ridged microfluidic channel translates each cell perpendicular to the channel axis in proportion to its stiffness. We demonstrate the physical principle of the cell sorting mechanism and show that our microfluidic approach can be effectively used to separate a variety of cell types which are similar in size but of different stiffnesses, spanning a range from 210 Pa to 23 kPa. Atomic force microscopy is used to directly measure the stiffness of the separated cells and we found that the trajectories in the microchannel correlated to stiffness. We have demonstrated that the current processing throughput is 250 cells per second. This microfluidic separation technique opens new ways for conducting rapid and low-cost cell analysis and disease diagnostics through biophysical markers.

Leukocytes normally marginate toward the vascular wall in large vessels and within the microvasculature. Reversal of this process, leukocyte demargination, leads to substantial increases in the clinical white blood cell and granulocyte count and is a welldocumented effect of glucocorticoid and catecholamine hormones, although the underlying mechanisms remain unclear. Here we show that alterations in granulocyte mechanical properties are the driving force behind glucocorticoid-and catecholamine-induced demargination. First, we found that the proportions of granulocytes from healthy human subjects that traversed and demarginated from microfluidic models of capillary beds and veins, respectively, increased after the subjects ingested glucocorticoids. Also, we show that glucocorticoid and catecholamine exposure reorganizes cellular cortical actin, significantly reducing granulocyte stiffness, as measured with atomic force microscopy. Furthermore, using simple kinetic theory computational modeling, we found that this reduction in stiffness alone is sufficient to cause granulocyte demargination. Taken together, our findings reveal a biomechanical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination. In addition, in a broader sense, we have discovered a temporally and energetically efficient mechanism in which the innate immune system can simply alter leukocyte stiffness to fine tune margination/demargination and therefore leukocyte trafficking in general. These observations have broad clinically relevant implications for the inflammatory process overall as well as hematopoietic stem cell mobilization and homing. cellular mechanics | leukocyte deformability | demargination | microfluidics | atomic force microscopy L eukocyte margination within the microvasculature and in larger blood vessels is an integral part of the inflammatory process and innate immune system (1, 2). This margination phenomenon is twofold, involving sequestration of leukocytes in the capillary bed (3, 4) as well as movement of leukocytes toward the blood vessel wall (Fig. 1A) (5, 6). Recent experimental and computational data, including our own, indicate that the mechanical properties of leukocytes play a major role in margination and are sufficient to drive leukocytes in whole blood toward the vessel wall (7-12).What is not known is whether leukocyte softening can cause the reversal of leukocyte margination, which would indicate that leukocyte stiffness may be modulated by the immune system as an additional biophysical means to mediate leukocyte trafficking. To that end, we explored whether leukocyte stiffness alterations play a role in leukocyte demargination induced by glucocorticoid and catecholamine hormones. Although this phenomenon, which causes significant increases in the white blood cell (WBC) count within the clinical complete blood count (CBC) and specifically involves the granulocyte subpopulation of leukocytes, has been well documented from a clinical perspective for deca...

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