Cellular softening mediates leukocyte demargination and trafficking, thereby increasing clinical blood counts.

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 well-documented 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 enrichment through microfluidic fractionation based on cell biomechanical properties.

The biomechanical properties of populations of diseased cells are shown to have differences from healthy populations of cells, yet the overlap of these biomechanical properties can limit their use in disease cell enrichment and detection. We report a new microfluidic cell enrichment technology that continuously fractionates cells through differences in biomechanical properties, resulting in highly pure cellular subpopulations. Cell fractionation is achieved in a microfluidic channel with an array of diagonal ridges that are designed to segregate biomechanically distinct cells to different locations in the channel. Due to the imposition of elastic and viscous forces during cellular compression, which are a function of cell biomechanical properties including size and viscoelasticity, larger, stiffer and less viscos cells migrate parallel to the diagonal ridges and exhibit positive lateral displacement. On the other hand, smaller, softer and more viscous cells migrate perpendicular to the diagonal ridges due to circulatory flow induced by the ridges and result in negative lateral displacement. Multiple outlets are then utilized to collect cells with finer gradation of differences in cell biomechanical properties. The result is that cell fractionation dramatically improves cell separation efficiency compared to binary outputs and enables the measurement of subtle biomechanical differences within a single cell type. As a proof-of-concept demonstration, we mix two different leukemia cell lines (K562 and HL60) and utilize cell fractionation to achieve over 45-fold enhancement of cell populations, with high purity cellular enrichment (90% to 99%) of each cell line. In addition, we demonstrate cell fractionation of a single cell type (K562 cells) into subpopulations and characterize the variations of biomechanical properties of the separated cells with atomic force microscopy. These results will be beneficial to obtaining label-free separation of cellular mixtures, or to better investigate the origins of biomechanical differences in a single cell type.

Microfluidic cellular enrichment and separation through differences in viscoelastic deformation.

We report a microfluidic approach to separate and enrich a mixture of two cell types based on differences in cell viscoelastic behavior during repeated compressions and relaxation events. As proof of concept, we demonstrate that variations in viscoelasticity affect the flow trajectory of one type of leukemia cell line (K562) in relation to another leukemia cell line (HL60) as well as healthy leukocytes. These differences in cell trajectory can be utilized to enrich and sort K562 cells from HL60 cells and leukocytes. The microfluidic device utilizes periodic, diagonal ridges to compress and translate the cells laterally perpendicular to channel axis. The ridge spacing is tuned to allow relaxation of the K562 cells but not the HL60 cells or leukocytes. Therefore, the periodic compression laterally translates weakly viscous cells, while highly viscous cells respond to hydrodynamic circulation forces generated by the slanted ridges. As a result, cell sorting has strong dependency on cell viscosity. We use atomic force microscopy and high-speed optical microscopy to measure cell stiffness, cell relaxation rate constant, and cell size for all cell types. With properly designed microfluidic channels, we can optimize the enrichment of K562 cells from HL60 and leukocytes.