Self-assembled and perfusable microvasculature-on-chip for modeling leukocyte trafficking.

Leukocyte recruitment from blood to tissue is a process that occurs at the level of capillary vessels during both physiological and pathological conditions. This process is also relevant for evaluating novel adoptive cell therapies, in which the trafficking of therapeutic cells such as chimeric antigen receptor (CAR)-T cells throughout the capillaries of solid tumors is important. Local variations in blood flow, mural cell concentration, and tissue stiffness contribute to the regulation of capillary vascular permeability and leukocyte trafficking throughout the capillary microvasculature. We developed a platform to mimic a biologically functional human arteriole-venule microcirculation system consisting of pericytes (PCs) and arterial and venous primary endothelial cells (ECs) embedded within a hydrogel, which self-assembles into a perfusable, heterogeneous microvasculature. Our device shows a preferential association of PCs with arterial ECs that drives the flow-dependent formation of microvasculature networks. We show that PCs stimulate basement membrane matrix synthesis, which affects both vessel diameter and permeability in a manner correlating with the ratio of ECs to PCs. Moreover, we demonstrate that hydrogel concentration can affect capillary morphology but has no observed effect on vascular permeability. The biological function of our capillary network was demonstrated using an inflammation model, where significantly higher expression of cytokines, chemokines, and adhesion molecules was observed after tumor necrosis factor-alpha (TNF-α) treatment. Accordingly, T cell adherence and transendothelial migration were significantly increased in the immune-activated state. Taken together, our platform allows the generation of a perfusable microvasculature that recapitulates the structure and function of an in vivo capillary bed that can be used as a model for developing potential immunotherapies.

Scalable Production of Monodisperse Bioactive Spider Silk Nanowires.

Elongated protein-based micro- and nanostructures are of great interest for a wide range of biomedical applications, where they can serve as a backbone for surface functionalization and as vehicles for drug delivery. Current production methods for protein constructs lack precise control of either shape and dimensions or render structures fixed to substrates. This work demonstrates production of recombinant spider silk nanowires suspended in solution, starting with liquid bridge induced assembly (LBIA) on a substrate, followed by release using ultrasonication, and concentration by centrifugation. The significance of this method lies in that it provides i) reproducability (standard deviation of length <13% and of diameter <38%), ii) scalability of fabrication, iii) compatibility with autoclavation with retained shape and function, iv) retention of bioactivity, and v) easy functionalization both pre- and post-formation. This work demonstrates how altering the function and nanotopography of a surface by nanowire coating supports the attachment and growth of human mesenchymal stem cells (hMSCs). Cell compatibility is further studied through integration of nanowires during aggregate formation of hMSCs and the breast cancer cell line MCF7. The herein-presented industrial-compatible process enables silk nanowires for use as functionalizing agents in a variety of cell culture applications and medical research.