Microfluidic cell adhesion assays have emerged as a means to increase throughput as well as reduce the amount of costly reagents. However as dimensions of the flow chamber are reduced and approach the diameter of a cell (D(c)), theoretical models have predicted that mechanical stress, force, and torque on a cell will be amplified. We fabricated a series of microfluidic devices that have a constant width:height ratio (10:1) but with varying heights. The smallest microfluidic device (200 μm ×20 μm) requires perfusion rates as low as 40 nL/min to generate wall shear stresses of 0.5 dynes/cm(2). When neutrophils were perfused through P-selectin coated chambers at equivalent wall shear stress, rolling velocities decreased by approximately 70 % as the ratio of cell diameter to chamber height (D(c)/H) increased from 0.08 (H = 100 μm) to 0.40 (H = 20 μm). Three-dimensional numerical simulations of neutrophil rolling in channels of different heights showed a similar trend. Complementary studies with PSGL-1 coated microspheres and paraformaldehyde-fixed neutrophils suggested that changes in rolling velocity were related to cell deformability. Using interference reflection microscopy, we observed increases in neutrophil contact area with increasing chamber height (9-33 %) and increasing wall shear stress (28-56 %). Our results suggest that rolling velocity is dependent not only on wall shear stress but also on the shear stress gradient experienced by the rolling cell. These results point to the D(c)/H ratio as an important design parameter of leukocyte microfluidic assays, and should be applicable to rolling assays that involve other cell types such as platelets or cancer cells.
Traditional leukocyte adhesion assays have provided significant insight into the mechanisms of leukocyte rolling in part through the use of homogeneously coated surfaces. These assays typically involve protein coating of glass coverslips or plastic petri dishes applied via a static drop of protein solution. With this approach, it is difficult to spatially control the location of proteins to fabricate surface-bound protein gradients that mimic in vivo situations. Microfluidic patterning of proteins with microfluidic devices has become a popular technique due to the ability to spatially pattern proteins on a cellular scale. Despite the advantages of microfluidic patterning, few studies have systematically investigated the effects of perfusion time, protein concentration, and perfusion shear stress on protein deposition. Herein, we demonstrated the fabrication of both line and step gradients of P-selectin on glass substrates that support cell rolling and adhesion assays. Investigation of the flow conditions during the microfluidic patterning led to several significant findings. We observed that the protein deposition time of 5 min was sufficient to deposit adequate P-selectin to support neutrophil rolling. We demonstrated that the amount of membrane P-selectin (mP-selectin) or recombinant P-selectin (rP-selectin) deposited showed a dependence on the perfusion shear stress between 4.0 and 32.0 dyn/cm(2), while similar studies with fibronectin or fibrinogen showed no shear stress dependence. Finally, we also created step changes in surface adherent protein concentration of P-selectin to characterize leukocyte-rolling behavior in response to sudden changes in ligand density.
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