Let's Get Rolling: Precise Control of Microfluidic Assay Conditions to Recapitulate Selectin-Mediated Rolling Interactions of the Leukocyte Adhesion Cascade.

The leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl-LewisX (sLeX) and endothelial E- and P-selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and "roll" on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step-by-step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin-mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing dishes for cell rolling experiments Basic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experiments Alternate Protocol 1: Protein conjugation with N6 linker Alternate Protocol 2: HUVEC culturing for monolayers Basic Protocol 3: Conducting cell rolling experiments on polyacrylamide gels Basic Protocol 4: ImageJ analysis of cell rolling movies Basic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras).

Elimination of Induced Hypoxic Regions in Depth of 3D Porous Silk Scaffolds by the Introduction of Channel Configuration.

Development of large, clinically sized tissue constructs with efficient mass transport is a tremendous need in tissue engineering. One major challenge in large tissue-engineered constructs is to support homogeneous delivery of oxygen and nutrients throughout the tissue scaffold while eliminating induced hypoxic regions in depth. To address this goal, we introduced an especial channeled architecture on porous silk-based tissue scaffolds to improve supplying of oxygen to the cells in central regions of the scaffolds. Oxygen gradients were measured and evaluated in three scaffold prototypes, namely, one unchanneled and two channeled scaffolds with different channel diameters (500 µm and 1000 µm). The channels were introduced into the constructs using stainless-steel rods arranged uniformly in stainless-steel mold, a fabrication method that enables precise control over channel diameter and the distance between channels. During 2-week culture of G292 cells, the 1000 µm channeled scaffolds demonstrated higher oxygen concentration at the center compared to 500 µm channeled prototype; however, the oxygen concentration approached the same level around the last days of culture. Nevertheless, homogenous oxygen distribution throughout the 1000 µm channeled constructs and the consequence of higher cell proliferation at day 14 postseeding corroborate the efficient elimination of induced hypoxic regions; and therefore, it holds promise for clinically relevant sized scaffold especially in bone tissue engineering.