An integrated assay to probe endothelial glycocalyx-blood cell interactions under flow in mechanically and biochemically well-defined environments

Abstract

Cell-cell and cell-glycocalyx interactions under flow are important for the behaviour of circulating cells in blood and lymphatic vessels. However, such interactions are not well understood due in part to a lack of tools to study them in defined environments. Here, we develop a versatile in vitro platform for the study of cell-glycocalyx interactions in well-defined physical and chemical settings under flow. Our approach is demonstrated with the interaction between hyaluronan (HA, a key component of the endothelial glycocalyx) and its cell receptor CD44. We generate HA brushes in situ within a microfluidic device, and demonstrate the tuning of their physical (thickness and softness) and chemical (density of CD44 binding sites) properties using characterisation with reflection interference contrast microscopy (RICM) and application of polymer theory. We highlight the interactions of HA brushes with CD44-displaying beads and cells under flow. Observations of CD44+ beads on a HA brush with RICM enabled the 3-dimensional trajectories to be generated, and revealed interactions in the form of stop and go phases with reduced rolling velocity and reduced distance between the bead and the HA brush, compared to uncoated beads. Combined RICM and bright-field microscopy of CD44+ AKR1 T-lymphocytes revealed complementary information about the dynamics of cell rolling and cell morphology, and highlighted the formation of tethers and slings, as they interacted with a HA brush under flow. This platform can readily incorporate more complex models of the glycocalyx, and should permit the study of how mechanical and biochemical factors are orchestrated to enable highly selective blood cell-vessel wall interactions under flow.

Self-regenerating giant hyaluronan polymer brushes

“…In summary, MEF cells flourish within the brush environment. These preliminary findings demonstrate that the HA brushes will be a useful platform to systematically study the interplay of sugar-rich hydrogels and glycocalyx in cell adhesion, an area of growing relevance and interest 62–66 .…”

A Method to Quantify Molecular Diffusion within Thin Solvated Polymer Films: A Case Study on Films of Natively Unfolded Nucleoporins

“…This provided a high contrast image of the circular interference pattern (Newtonian rings) with minimal stray light. RIC micrographs with the interference patterns were analyzed with a custom-written algorithm implemented in LabView (described previously 51 ) to quantify the effective height at the center of the plane–sphere interface. Data were fitted over an area of 170 × 170 μm 2 typically encompassing six full interference fringe rings.…”

Blood cell - vessel wall interactions probed by reflection interference contrast microscopy

“…In particular, endothelial cells lining the blood vessels are covered with a thick and highly deformable polymer layer called glycocalyx that is first encountered by flowing cells when interacting with the vessel wall. To decipher the essential parameters controlling cell homing or repulsion, we have developed an in vitro platform allowing precise control of the surface functionalization (by a glycopolymer brush) mimicking the physiological glycocalyx and of the flow of cell mimetics that can display specific receptors of glycocalyx components, while permiting direct imaging of these mimetics at high speed [3]. Based on this platform, we have studied how the interplay between mechanical properties of the brush and specific biochemical interactions regulate cell-surface interactions under flow.…”

“…(e), analysing the mean square displacement of Brownian beads without flows permits quantifying the precision of in-plane localization as the offset of the curve (here ~5 nm rms). Adapted from[3].…”