In vitro blood-brain barrier (BBB) models have played an important role in studying processes such as immune cell trafficking and drug delivery, as well as contributing to the understanding of mechanisms of disease progression. Many biological and pathological processes in the cerebrovasculature occur in capillaries and hence the lack of robust hierarchical models at the capillary scale is a major roadblock in BBB research. Here, a double-templating technique for engineering hierarchical BBB models with physiological barrier function at the capillary scale is reported. First, the formation of hierarchical vascular networks using human umbilical vein endothelial cells is demonstrated. Then, barrier function is characterized in a BBB model using brain microvascular endothelial-like cells differentiated from induced pluripotent stem cells. Finally, immune cell adhesion and transmigration are characterized in response to perfusion with the inflammatory cytokine tumor necrosis factor-alpha, and it is shown that capillary-scale effects, such as leukocyte plugging, observed in mouse models, can be recapitulated. The double-templated hierarchical model enables the study of a wide range of biological and pathological processes related to the human BBB.

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Oxidative stress is a shared pathology of neurodegenerative disease and brain injuries, and is derived from perturbations to normal cell processes by aging or environmental factors such as UV exposure and air pollution. As oxidative cues are often present in systemic circulation, the blood–brain barrier (BBB) plays a key role in mediating the effect of these cues on brain dysfunction. Therefore, oxidative damage and disruption of the BBB is an emergent focus of neurodegenerative disease etiology and progression. We assessed barrier dysfunction in response to chronic and acute oxidative stress in 2D and 3D in vitro models of the BBB with human iPSC-derived brain microvascular endothelial-like cells (iBMECs). We first established doses of hydrogen peroxide to induce chronic damage (modeling aging and neurodegenerative disease) and acute damage (modeling the response to traumatic brain injury) by assessing barrier function via transendothelial electrical resistance in 2D iBMEC monolayers and permeability and monolayer integrity in 3D tissue-engineered iBMEC microvessels. Following application of these chronic and acute doses in our in vitro models, we found local, discrete structural changes were the most prevalent responses (rather than global barrier loss). Additionally, we validated unique functional changes in response to oxidative stress, including dysfunctional cell turnover dynamics and immune cell adhesion that were consistent with changes in gene expression.

Lyme disease is a tick‐borne disease prevalent in North America, Europe, and Asia. Despite the accumulated knowledge from epidemiological, in vitro, and in animal studies, the understanding of dissemination of vector‐borne pathogens, such as Borrelia burgdorferi (Bb), remains incomplete with several important knowledge gaps, especially related to invasion and intravasation into circulation. To elucidate the mechanistic details of these processes a tissue‐engineered human dermal microvessel model is developed. Fluorescently labeled Bb are injected into the extracellular matrix (ECM) to mimic tick inoculation. High resolution, confocal imaging is performed to visualize the sub‐acute phase of infection. From analysis of migration paths no evidence to support adhesin‐mediated interactions between Bb and ECM components is found, suggesting that collagen fibers serve as inert obstacles to migration. Intravasation occurs at cell–cell junctions and is relatively fast, consistent with Bb swimming in ECM. In addition, it is found that Bb alone can induce endothelium activation, resulting in increased immune cell adhesion but no changes in global or local permeability. Together these results provide new insight into the minimum requirements for Bb dissemination and highlight how tissue‐engineered models are complementary to animal models in visualizing dynamic processes associated with vector‐borne pathogens.

Lyme disease is a tick-borne disease prevalent in North America, Europe, and Asia. Dissemination of vector-borne pathogens, such as Borrelia burgdorferi (Bb), results in infection of distant tissues and is the main contributor to poor outcomes. Despite the accumulated knowledge from epidemiological, in vitro, and in animal studies, the understanding of dissemination remains incomplete with several important knowledge gaps, especially related to invasion and intravasation at the site of a tick bite, which cannot be readily studied in animal models or humans. To elucidate the mechanistic details of these processes we developed a tissue-engineered human dermal microvessel model. Fluorescently-labeled Bb (B31 strain) were injected into the extracellular matrix (ECM) of the model to mimic tick inoculation. High resolution, confocal imaging was performed to visualize Bb migration in the ECM and intravasation into circulation. From analysis of migration paths we found no evidence to support adhesin-mediated interactions between Bb and components of the ECM or basement membrane, suggesting that collagen fibers serve as inert obstacles to migration. Transendothelial migration occurred at cell-cell junctions and was relatively fast, consistent with Bbswimming in ECM. In addition, we found that Bb alone can induce endothelium activation, resulting in increased immune cell adhesion but no changes in global or local permeability. Together these results provide new insight into the minimum requirements for dissemination of Bb at the site of a tick bite, and highlight how tissue-engineered models are complementary to animal models in visualizing dynamic processes associated with vector-borne pathogens.