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The blood brain barrier (BBB) is a complex, multi-cellular structure that strictly regulates the passage of blood-borne molecules, ions, and cells from the periphery into the central nervous system (CNS). Its constituents include specialized endothelial cells (EC), pericytes, astrocytic end-feet, and the basal lamina. Due to this protective structure, hydrophilic drugs suboptimally accumulate in the brain after systemic administration contributing to the clinical failure of many CNS targeted therapies. Moreover, BBB dysfunction is observed in many neurodegenerative illnesses including Parkinson’s, Alzheimer’s, and Huntington’s disease. Thus, there is considerable interest in elucidating the underpinnings of BBB transport such that endogenous systems may be modulated or coopted to best treat CNS-associated diseases.While maintained by exogenous factors, BBB properties are localized to and effectuated by the brain endothelium. Unlike ECs of the periphery, brain microvascular ECs (BMECs) maintain specialized tight junctions between single cells that restrict paracellular entry into the CNS, while a limited number of caveolae, reduced pinocytosis, and selective transporters limit transcellular entry. This specialization, induced by the developing neural milieu and maintained by non-endothelial brain constituents, is critical for BMEC identity. Consequently, there is a need for higher-order human-derived BBB in vitro models to replace conventional BMEC monolayer models that are better suited to complement in vivo (mouse) studies.Based on our lab’s Vascularized Micro Organ (VMO) platform, this body of research aims to develop a novel BBB version – the Vascularized Micro Brain (VMB) – utilizing human-derived endothelial colony forming cells derived endothelial cells (ECFC-ECs), pericytes, astrocytes and iPSC-derived neural progenitor cells (iPSC-NPCs), that mimics the natural process of BBB induction and assembles into a perfused micro-vasculature. Herein, we demonstrate the ability of the VMB to mirror in vivo BBB gene expression and capture complex BBB-like vascular functions including reduced permeability and transcytosis. We believe that the VMB is optimally suited to study the BBB and its functional state during CNS-related pathologies, including neurodegenerative diseases and cancer, with the hope that it can better identify drugs likely to succeed in the clinic.

Metastasis remains the leading cause of cancer mortality but its mitochondrial dysregulation - from its morphology and motility to its metabolic influence - in modulating cancer’s progression is poorly understood. This dissertation describes the development and use of complex microphysiological culture systems to place cancer in its proper environmental context for mitochondrial phenotypic discoveries. FLIM of NADH is used to probe the metabolic differences between invasive and non- or less-invasive cancer cells and elucidate potential invasion-specific therapeutic targets. This dissertation also introduces Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer finds that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, Mitometer shows that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is then applied to investigate cellular contractility in the influence of mitochondrial phenotype. The goal of this dissertation as a whole is to accentuate the importance of creating new assays and techniques with the impact of context and translatability in mind.