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Hemostasis is an innate protective mechanism that plays a central role in maintaining the homeostasis of the vascular system during vascular injury. Studying this essential physiological process is often challenged by the difficulty of modeling and probing the complex dynamics of hemostatic responses in the native context of human blood vessels. To address this major challenge, this paper describes a microengineering approach for in vitro modeling of hemostasis. Our microphysiological model replicates the living endothelium, multilayered microarchitecture, and procoagulant activity of human blood vessels, and is also equipped with a microneedle that can be actuated with spatial precision to simulate penetrating vascular injuries. The system recapitulates key features of the hemostatic response to acute vascular injury as observed in vivo, including i) thrombin-driven accumulation of platelets and fibrin, ii) formation of a platelet- and fibrin-rich hemostatic plug that halts blood loss, and iii) matrix deformation driven by platelet contraction for wound closure. Moreover, the potential use of this model for drug testing applications is demonstrated by evaluating the effects of anticoagulants and antiplatelet agents that are in current clinical use. The vascular injury-on-a-chip may serve as an enabling platform for preclinical investigation of hematological disorders and emerging therapeutic approaches against them.

T cells navigate a wide variety of tissues and organs for immune surveillance and effector functions. Although nanoscale topographical structures of extracellular matrices and stromal/endothelial cell surfaces in local tissues may guide the migration of T cells, there has been little opportunity to study how nanoscale topographical features affect T cell migration. In this study, we systematically investigated mechanisms of nanotopography-guided migration of T cells using nanoscale ridge/groove surfaces. The velocity and directionality of T cells on these nanostructured surfaces were quantitatively assessed with and without confinement, which is a key property of three-dimensional interstitial tissue spaces for leukocyte motility. Depending on the confinement, T cells exhibited different mechanisms for nanotopography-guided migration. Without confinement, actin polymerization-driven leading edge protrusion was guided toward the direction of nanogrooves via integrin-mediated adhesion. In contrast, T cells under confinement appeared to migrate along the direction of nanogrooves purely by mechanical effects, and integrin-mediated adhesion was dispensable. Therefore, surface nanotopography may play a prominent role in generating migratory patterns for T cells. Because the majority of cells in periphery migrate along the topography of extracellular matrices with much lower motility than T cells, nanotopography-guided migration of T cells would be an important strategy to efficiently perform cell-mediated immune responses by increasing chances of encountering other cells within a given amount of time.

Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to be used for tissue engineering and regenerative medicine. Repairing nerve injury by differentiating hESCs into a neuronal lineage is one important application of hESCs. Biochemical and biological agents are widely used to induce hESC differentiation. However, it would be better if we could induce differentiation of hESCs without such agents because these factors are expensive and it is difficult to control the optimal concentrations for efficient differentiation with reduced side effects. Moreover, the mechanism of differentiation induced by these factors is still not fully understood. In this study, we present evidence that nanoscale ridge/groove pattern arrays alone can effectively and rapidly induce the differentiation of hESCs into a neuronal lineage without the use any differentiation-inducing agents. Using UV-assisted capillary force lithography, we constructed nanoscale ridge/groove pattern arrays with a dimension and alignment that were finely controlled over a large area. Human embryonic stem cells seeded onto the 350-nm ridge/groove pattern arrays differentiated into neuronal lineage after five days, in the absence differentiation-inducing agents. This nanoscale technique could be used for a new neuronal differentiation protocol of hESCs and may also be useful for nanostructured scaffolding for nerve injury repair.

The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely on the size of molecules, suggesting that molecular size is the major factor that influences tumor uptake by its effect on systemic clearance and diffusion into tumor. Affinity to tumor antigen failed to augment tumor uptake of Fab above non-specific accumulation, which suggests that Fab fragments of typical monoclonal antibodies may fall below an affinity threshold for use as molecular imaging agents. Despite abundant localization into the tumor, albumin and streptavidin were not found on cell surface or inside cells. By comparing biomolecules differing in size and affinity, our study highlights that while pharmacokinetics are a dominant factor in tumor uptake for biomolecules, affinity to tumor antigen is required for tumor binding and internalization.

We present a simple receding meniscus induced method to capture non-adherent yeast cells onto microwells inside microfluidic channels. Microwells were fabricated by capillary molding onto glass substrate using a UV curable polyurethane acrylate (PUA) solution, leading to well-defined, robust microstructures. A cell suspension of the budding yeast, Saccharomyces cerevisiae, was introduced into microfluidic channels by capillary filling and a receding meniscus was subsequently generated by evaporation. As the meniscus receded, yeast cells were spontaneously captured onto microwells at single-cell level. Using this cell-based platform, we observed the response of yeast cells upon stimulation by a mating pheromone (alpha-factor) by monitoring the expression of green fluorescent protein (GFP) with time. It was observed that alpha-factor triggered the expression of GFP at 60 min after stimulation and the fluorescence intensity was sustained for additional 60 min without changes.

We present a simple cell docking method induced by receding meniscus to capture non-adherent yeast cells onto microwells inside a microfluidic channel. Microwells were fabricated either by capillary moulding of UV curable polyurethane acrylate (PUA) onto glass substrate or direct replica moulding of poly(dimethyl siloxane) (PDMS). A cell suspension of the budding yeast, Saccharomyces cerevisiae, was introduced into the microfluidic channel by surface tension driven capillary flow and a receding meniscus was subsequently generated by evaporation. As the meniscus progressed, one to multiple yeast cells were spontaneously captured onto microwells by lateral capillary force created at the bottom of the meniscus. Using this cell-based platform, we observed the response of yeast cells upon stimulation by a mating pheromone (alpha-factor) by monitoring the expression of green fluorescent protein (GFP) with time. It was observed that alpha-factor triggered the expression of GFP at 60 min after stimulation and the fluorescence intensity was sustained for an additional 60 min without changes.

We present a simple receding meniscus induced method to capture non-adherent yeast cells onto microwells inside microfluidic channels. Microwells were fabricated by capillary molding onto glass substrate using a UV curable polyurethane acrylate (PUA) solution, leading to well-defined, robust microstructures. A cell suspension of the budding yeast, Saccharomyces cerevisiae, was introduced into microfluidic channels by capillary filling and a receding meniscus was subsequently generated by evaporation. As the meniscus receded, one to five yeast cells were spontaneously captured onto microwells by lateral capillary force created at the thin region of the meniscus. Using this cell-based platform, we observed the response of yeast cells upon stimulation by a mating pheromone (alpha-factor) by monitoring the expression of green fluorescent protein (GFP) with time. It was observed that alpha-factor triggered the expression of GFP at 60 min after stimulation and the fluorescence intensity was sustained for additional 60 min without changes

Intraluminal crawling is considered to be important for extravasation of leukocytes in blood vessels, but biochemical/biophysical cues guiding the crawling of leukocytes have not been clearly understood. Here we provide evidence that T cells sense the topography of luminal surfaces and the nuclei of endothelial cells (ECs) using lamellipodia and filopodia, respectively, to optimize path finding during intraluminal crawling. Well-aligned EC layers or replicas of EC layers, which exhibit topography similar to that of EC layers, were fabricated, and flow was applied either parallel or perpendicular to the orientation of EC alignment. T cells crawled along the valleys of the topographical landscapes of the EC layers, while avoiding nuclei of ECs regardless of flow direction. Pharmacological inhibitor treatments revealed that sensing of topography and nuclei of EC layers was mediated by lamellipodia and filopodia, respectively. Lamellipodia or filopodia-inhibited T cells crawled significantly longer distances for extravasation than did normal T cells, indicating that sensing biophysical cues are critical for optimizing routes for extravasation.