Effects of shear on P-selectin deposition in microfluidic channels

Abstract

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.

An In Vitro Model for Assessing Corneal Keratocyte Spreading and Migration on Aligned Fibrillar Collagen

“…Microfluidic devices were fabricated in polydimethylsiloxane (PDMS) as previously described [42]. Briefly, a standard negative photolithography procedure was utilized to fabricate a straight channel photoresist template (height = 60 µm, width = 1500 µm, length = 22,000 µm) in KMPR 1050 photoresist (Micr Chem, Westborough, MA, USA).…”

Nox2 Regulates Platelet Activation and NET Formation in the Lung

“…The microfluidic channels had a cross-sectional dimension of 300 μm wide by 50 μm tall and were fabricated by standard photolithography procedures described previously (23, 24). To measure the frequency of neutrophil-platelet aggregates, the microfluidic chambers were initially filled with Hanks' balanced salt solution and then coated with recombinant mouse P-selectin by perfusing a 0.2 μg/ml P-selectin solution through the channel at 1,600 s −1 for 15 min (25). The microfluidic channels were then blocked by filling the channels with a solution of 0.5% human serum albumin in Hanks' balanced salt solution and incubating for 30 min at room temperature before placing the microfluidic device on a Zeiss AxioVert A1 microscope equipped with a 63 × Plan-Apochromat (NA 1.4) objective and a Hamamatsu ORCA-Flash 4.0 scientific CMOS camera.…”

Effects of Transient Exposure to High Shear on Neutrophil Rolling Behavior

“…Microfluidic devices were prepared from poly-dimethyl siloxane (PDMS; Dow Corning, Midland, MI) in a 10:1 ratio as previously described. 8,44 Briefly, the master mold was fabricated through a standard negative photolithography technique using KMPR 1050 photoresist (MicroChem, Westborough, MA). The dimensions of the different microfluidic channels fabricated are listed in Figure 1.…”