Endothelial cells (ECs) line the microvasculature and constitute a barrier between the vessel lumen and surrounding tissues. ECs inform circulating immune cells of the health and integrity of surrounding tissues, recruiting them in response to pathogens and tissue damage. ECs play an active role in the transmigration of immune cells across the vessel wall. We have discovered important differences in the properties of ECs on soft hydrogel substrates of varying stiffness, in comparison to glass. Primary ECs from several human sources were tested; all formed monolayers normally on soft substrates. EC monolayers formed more mature cell-cell junctions on soft substrates, relative to glass, based on increased recruitment of vinculin and F-actin. EC monolayers supported transendothelial migration (TEM) on soft substrates. Immune cells, including peripheral blood lymphocytes (PBLs) and natural killer cells, showed decreasing numbers of paracellular (between-cell) transmigration events with decreasing substrate stiffness, while the number of transcellular (through-cell) events increased for PBLs. Melanoma cancer cells showed increased transmigration with decreased stiffness. Our findings demonstrate that endothelial monolayers respond to the mechanical properties of their surroundings, which can regulate the integrity and function of the monolayer independently from inflammatory signals. Soft hydrogel substrates are a more appropriate and physiological model for tissue environments than hard substrates, with important implications for the experimental analysis of

Natural Killer (NK) cells perform many functions that depend on actin assembly, including adhesion, chemotaxis, lytic synapse assembly and cytolysis. HS1, the hematopoietic homolog of cortactin, binds to Arp2/3 complex and promotes actin assembly by helping to form and stabilize actin filament branches. We investigated the role of HS1 in transendothelial migration (TEM) by NK cells. Depletion of HS1 led to a decrease in the efficiency of TEM by NK cells, as measured by transwell assays with endothelial cell monolayers on porous filters. Transwell assays involve chemotaxis of NK cells across the filter, so to examine TEM more specifically, we imaged live-cell preparations and antibody-stained fixed preparations, with and without the chemoattractant SDF-1α. We found small to moderate effects of HS1 depletion on TEM, including whether the NK cells migrated via the transcellular or paracellular route. Expression of HS1 mutants indicated that phosphorylation of HS1 tyrosines at positions 222, 378 and 397 was required for rescue in the transwell assay, but HS1 mutations affecting interaction with Arp2/3 complex or SH3-domain ligands had no effect. The GEF Vav1, a ligand of HS1 phosphotyrosine, influenced NK cell transendothelial migration. HS1 and Vav1 also affected the speed of NK cells migrating across the surface of the endothelium. We conclude that HS1 has a role in transendothelial migration of NK cells and that HS1 tyrosine phosphorylation may signal through Vav1.

Insulin-producing human embryonic stem cell-derived β (SC-β) cells are a promising cell source for diabetes cell replacement therapy. We have recently reported a differentiation strategy that produces SC-β cells in islet organoids that not only undergo glucose-stimulated insulin secretion but also have an islet-like dynamic insulin release profile, displaying both first and second phase insulin secretion. The goal of this study was to further characterize the functional profile of these SC-β cells in vitro. We utilized a Seahorse extracellular flux analyzer to measure mitochondrial respiration of SC-β cells at low and high glucose. We also used photolithography to fabricate a microfluidic device containing microwells to immobilize SC-β cells for perfusional analysis, monitoring cytoplasmic calcium using Fluo-4 AM at low and high glucose. Here we find that in addition to increased insulin secretion, SC-β cells have increased cellular respiration and cytoplasmic calcium ion concentration in response to a high glucose stimulation. Our results indicate that SC-β cells have similar function to that reported for islets, providing further performance characterization that could help with eventual development for diabetes cell therapy and drug screening.3 Diabetes Mellitus (DM) is a group of metabolic disorders that leads to the inability of the body to regulate blood glucose levels. Type 1 diabetes (T1D) involves the autoimmunemediated destruction of the insulin-producing pancreatic β cells located in the islets of Langerhans, leading to insulin deficiency and hyperglycemia. T1D is typically managed by injection of exogenous insulin. However, this clinical intervention does not emulate the normal behavior of the native β cells, making patients at risk for many long term complications 1 . An alternative therapy for T1D is the transplantation of cadaveric human islets, with the hope of reducing hyper-and hypoglycemic episodes. Some patients who were transplanted with islets have remained insulin independent for several years 2 . A major limitation of this approach, however, is the scarcity and quality of islets sourced from cadavers, limiting the widespread application of this therapy. Differentiation of human embryonic stem cells (hESCs) to insulinproducing β (SC-β) cells in islet organoids could serve as an unlimited supply of cells to treat millions of patients 3 , particularly if combined with transplantation strategies that vascularization, allow retrievability, and/or protect the cells from immune attack 4-8 .We recently reported a strategy for making large numbers of SC-β cells from hESCs 9 . This protocol is highly efficient, generating an almost pure population of pancreatic endocrine, of which most cells express insulin. These cells are highly functional, capable of undergoing glucose-stimulated insulin secretion. These cells were capable of restoring glucose tolerance when transplanted into streptozotocin-treated mice. Of particular note was the ability of these cells to display first and second phase insulin s...

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A reduced order computational model and imaging experiments are presented as a combined method to investigate migration and trapping of microscale particles within an ultrasonic droplet generator. Use of two-dimensional (2D) cross-sectional representations of the three-dimensional (3D) device enables observation of acoustic focusing phenomena that are otherwise visually inaccessible. Our approach establishes relationships between system operating parameters and particle retention due to acoustic radiation forces that arise during atomization of heterogeneous particle suspensions. The droplet generator consists of a piezoelectric transducer for ultrasonic actuation, a resonant fluid-filled chamber, and an array of microscopic pyramidal nozzles. 2D visualization chips were produced through anodic bonding of glass to microfluidic reservoirs deep reactive ion etched in silicon. Open nozzle orifices of the 3D microarray were sealed in its 2D representation to facilitate filling and testing. Finite element analysis was used to model the harmonic response of the 2D assembly from 500 kHz to 2 MHz. The average nozzle tip pressure amplitude across the 2D array was then used to identify operating frequencies that correspond to optimal droplet ejection from the 3D device (ejection modes). The pressure field at these resonant frequencies predicts the equilibrium distribution of polymeric beads suspended in the reservoirs of the 2D chips. To qualitatively assess the accuracy of the model results, visualization experiments were performed at the first three ejection modes of the system (fn1 ≈ 620–680 kHz, fn2 ≈ 1.14 MHz, and fn3 ≈ 1.63 MHz) using 10 μm polystyrene beads. The model demonstrates a remarkable ability to capture the overall shape, as well as specific details of the terminal particle distributions, defined as the state with no further movement toward a pressure node or antinode. Finally, time course trials of acoustic focusing of heterogeneous particle suspensions were used to observe the influence of particle volume on the magnitude of the acoustic radiation force. A mixture of 5 μm and 20 μm diameter polystyrene beads was subjected to a standing acoustic field in the 2D chips. Particle position was recorded at 5 ms intervals until an equilibrium distribution was achieved. As expected, the larger beads focused much more rapidly than smaller beads, acquiring their final positions in seconds (versus 10s of seconds for the 5 μm particles). The method and results reported here serve as building blocks toward translation of an existing ultrasonic droplet generator into a high-throughput particle separation and isolation platform.