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Effective, rapid, and functionally perfusable vascularization remains a major challenge in tissue engineering. Current approaches to generating vasculature in vitro require multipart fabrication methods or complex and costly media supplements, limiting their scalability. Here, we demonstrate that exogenous electrical stimulation (ESTIM) offers a promising alternative by enhancing 3D vasculogenesis in engineered human tissues. Exposing 3D endothelial‐fibroblast cocultures to pulsed ESTIM promoted the formation of dense and branched vascular networks. In a microfluidic device model, we show that ESTIM induces the formation of a perfusable, interconnected vascular network, whereas unstimulated networks remain less mature. Importantly, we demonstrate that upon implantation, ESTIM‐pretreated vascular grafts exhibit elevated anastomosis with the host and perfusion with blood relative to the untreated grafts. In addition, we use ESTIM to promote engraftment of a vascularized 3D liver construct. Mechanistically, we find that ESTIM induces membrane hyperpolarization in endothelial cells via voltage‐gated potassium channels (KV). Inhibiting KVs abrogated ESTIM's pro‐vasculogenic effects in endothelial cells. Conversely, pharmacologically activating hyperpolarization induced endothelial responses even without ESTIM, directly linking KV‐mediated hyperpolarization as a key mechanism by which ESTIM drives vascular assembly and function. Ultimately, our work establishes ESTIM as a new orthogonal approach to promote the formation of perfusable vasculature in engineered tissues. Electrical stimulation (ESTIM) enhances vasculature formation in engineered human tissues. In 3D endothelial‐fibroblast constructs, conditioning with ESTIM promotes the formation of dense and highly branched networks that rapidly anastomose with mouse vasculature when implanted in vivo. Vessels formed under ESTIM are importantly functionally perfusable. Mechanistically, ESTIM induces endothelial membrane hyperpolarization via voltage‐gated potassium channels, linking bioelectric cues to improved vascularization.

Bile flow is an essential feature of the liver, and disruption of this process contributes to a range of liver pathologies. Efficient bile transport requires coordinated organization between hepatocytes and cholangiocytes at the hepatobiliary junction, a structure that remains poorly captured in existing in vitro models of liver disease. Here, we present a 3D multicellular spheroid‐based model of the human hepatobiliary junction. Building on advances in organoid and spheroid engineering, we co‐aggregate human hepatocytes and intrahepatic cholangiocytes, supported by murine fibroblasts, into adult hepatobiliary organoids (aHBOs). aHBOs directionally transport bile from hepatocyte canaliculi to cholangiocyte‐lined ductule‐like structures, visualized through a high‐throughput imaging assay. Hepatobiliary junction formation and bile flow dynamics are quantified over time using a fluorescent bile acid analog and AI‐assisted image analysis. When subjected to hypoxia‐reoxygenation, aHBOs exhibit disrupted bile transport and distinct cell‐type‐specific responses, enabling interrogation of hepatocyte and cholangiocyte vulnerability to transplant‐associated biliary hypoxia. Our findings suggest a reversible reduction in hepatocyte canalicular function under hypoxia, followed by selective cholangiocyte death upon reoxygenation, potentially contributing to biliary dysfunction after ischemic injury. This human‐derived, scalable platform provides a phenotypically relevant model for dissecting mechanisms of biliary dysfunction and discovering therapeutics for hypoxic liver injury and cholestatic diseases. A 3D human organoid platform reconstructs the hepatobiliary junction between primary adult hepatocytes and intrahepatic cholangiocytes. These adult hepatobiliary organoids (aHBOs) support directional bile transport from canaliculi to ductule‐like structures, enable quantitative imaging of junction dynamics, and reveal cell‐type‐specific vulnerabilities to hypoxia‐reoxygenation relevant to biliary injury and cholestatic disease.

Cholestasis, or disruption in bile flow, is a poorly-understood feature of many liver diseases and is a well-established indication for liver transplant. Despite this clinical significance, many tissue engineering strategies for modeling or treating liver disease fail to recapitulate physiological bile flow. Recent advances in the field of tissue engineering and organoid technology have enabled the culture of human hepatocytes and bile duct cells in vitro, these models lack a key function of the liver which is bile transport. In this thesis, I first describe developments in bioengineering technology that have allowed for the culture and manipulation of bile duct cell organoids. I then present a 3D multicellular spheroid model that captures the structure and function of the human hepatobiliary junction—the interface between liver and bile duct cells that is often disrupted in liver disease. By co-aggregating primary human hepatocytes and bile duct cells, I engineer a liver spheroid model that recapitulates physiological bile flow through a functional connection between the two cell types. These spheroids maintain cell polarity and transport bile from hepatocyte canaliculi to bile duct structures. This function is quantified by leveraging a high-throughput imaging assay with AI-assisted analysis to track junction formation and bile flow over time. I also use this system to model ischemic injury of the bile duct, a common complication of liver transplant, by tuning the oxygen parameters of the spheroid culture. In this injury model, I observe and describe two processes that potentially contribute to injury: a reversible loss of canalicular function during hypoxia, followed by selective bile duct cell death after reoxygenation. This human-based, scalable platform provides a new tool to study bile duct biology, understand mechanisms of biliary injury after liver transplant, and support drug discovery efforts for cholestatic liver diseases.

Effective, rapid and functionally perfusable vascularization remains a major challenge in tissue engineering. Current approaches to generate vasculature require multipart fabrication methods or complex and costly media supplements, limiting their scalability. Here, we demonstrate that exogenous electrical stimulation (estim) offers a promising alternative by enhancing 3D vasculogenesis in engineered human tissues. Exposing 3D endothelial-fibroblast cocultures to pulsed estim promoted the formation of dense and branched vascular networks. In a microfluidic device model, we show that estim induces the formation of an interconnected vascular network that can be perfused, whereas unstimulated control networks remained less mature. Importantly, we demonstrate that upon implantation, estim-pretreated vascular grafts exhibit elevated anastomosis with host and perfusion with blood relative to the untreated grafts. In addition, we use estim to promote engraftment of a vascularized 3D liver construct. Mechanistically, we find that estim induces membrane hyperpolarization in endothelial cells via voltage-gated potassium (K ) channels. Inhibiting K channels abrogated estim's pro-vasculogenic effects in endothelial cells. Conversely, pharmacologically activating hyperpolarization induced endothelial responses even without estim, directly linking K channel-mediated hyperpolarization as a key mechanism by which estim drives vascular assembly and function. Ultimately, our work establishes estim as a new orthogonal approach to promote formation of perfusable vasculature in engineered tissues.

Cholestasis, or disruption in bile flow, is a common yet poorly understood feature of many liver diseases and injuries. Despite this, many engineered human tissue models of liver disease fail to recapitulate physiological bile flow. Here, we present a 3D multicellular spheroid-based model of the human hepatobiliary junction, the interface between hepatocytes and cholangiocytes often disrupted in liver disease that is required for directing bile excreted by hepatocytes into the biliary ductal system. Building on advances in organoid and spheroid engineering, we co-aggregate human hepatocytes and intrahepatic cholangiocytes into adult hepatobiliary organoids (aHBOs) that structurally connect and functionally transport bile. aHBOs directionally transport bile from hepatocyte bile canaliculi to cholangiocyte-lined ductules, which we visualize through a high-throughput imaging assay. Hepatobiliary junction formation and bile flow dynamics are quantified over time using fluorescent bile acid analogs and AI-assisted image analysis. When subjected to hypoxia-reoxygenation, aHBOs recapitulate features of biliary dysfunction that mimics the cholestasis and ischemia-reperfusion injury that complicates liver transplant. Our findings suggest that 1) a reversible reduction in hepatocyte canalicular function under hypoxia, followed by 2) selective cholangiocyte death upon reoxygenation, are processes that potentially contribute to biliary dysfunction upon ischemic injury. This human-derived, scalable platform provides a phenotypically-relevant model for dissecting biliary pathophysiology and lays the groundwork for a therapeutic discovery platform for post-transplant ischemic cholangiopathy and other cholestatic liver diseases.

Liver transplantation remains the standard of care for end-stage liver failure, yet it is constrained by donor scarcity, surgical complexity, and restricted access for many patients. Cell-based therapies offer a potential alternative, yet their translation has been hindered by low engraftment, poor localization, and a lack of delivery strategies that are both effective and minimally invasive. To address these challenges, we developed a new approach termed INSITE (Injected Self-assembled Image-guided Tissue Ensembles), an injectable platform composed of primary human hepatocytes and hydrogel microspheres that can be delivered by image-guided injection and assembled in situ into supportive, vascularizable scaffolds. In vivo, ultrasound-guided delivery into an ectopic site enabled precise graft localization, persistent visibility under noninvasive imaging, and vascular integration. Hepatocytes within these niches remained confined to the scaffold and maintained long-term functional activity. Furthermore, tuning material properties allowed control over scaffold remodeling and vascular recruitment, providing a means to enhance graft function. By integrating image-guided delivery with a modular and supportive scaffold, INSITE establishes a clinically compatible strategy for advancing minimally invasive cell therapies.

Epithelial cancers disrupt tissue architecture and are often driven by mutations in genes that play important roles in normal epithelial morphogenesis. The intrahepatic biliary system is an epithelial tubular network that forms within the developing liver via the initiation and expansion of apical lumens. Intrahepatic biliary tumors (intrahepatic cholangiocarcinoma) commonly harbor activating genomic alterations in the FGFR2 receptor tyrosine kinase, which plays important roles in epithelial morphogenesis in other developmental settings. Using a physiologic and quantitative 3D model we demonstrate that FGFR signaling is important for biliary morphogenesis and that oncogenic FGFR2 fusions and in-frame deletions disrupt biliary architecture. Importantly, we show that the trafficking of and signaling from the FGFR2 mutants, as well as their phenotypic impacts, are governed by the epithelial state of the cell. Unexpectedly, we also found that distinct tumor-driving FGFR2 mutants disrupt biliary morphogenesis in completely different and clinically relevant ways, informing our understanding of morphogenesis and tumorigenesis and highlighting the importance of convergent studies of both.