Browsing by Author "Harrington, Daniel A"

Now showing 1 - 4 of 4
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    Directing Collective Epithelial Morphology Using a Light-Based Carving Tool
    (2022-08-11) Trubelja, Alen; Grande-Allen, Kathryn J; Harrington, Daniel A
    Head and Neck Cancer (HNC) refers to tumors that originate predominantly in the mouth, nose, and throat, accounting for more than 10,000 deaths yearly in the US. Radiotherapy is a common treatment for HNC. Patients that undergo radiotherapy (RT) oftentimes develop Xerostomia (dry mouth). This is an iatrogenic disorder with no cure, which significantly impacts quality of life. Xerostomia results from irreversible damage to the salivary glands (SG), which are complex branched organs with an unmet need for regenerative therapy. RT causes apoptosis of secretory salivary acinar cells and their progenitor source in the ducts. Tissue engineering could offer a therapeutic solution by harvesting healthy SG tissue prior to RT, expanding these cells in vitro to form 3D spheroids, then creating functional tissue for implantation post-RT. During development, salivary glands form by repeated cleft and bud formation, forming a divergent, duct-to-acini architecture difficult to recapitulate with standard gel scaffolds. Recent advances in biofabrication have enabled high-resolution control over user-defined architectures within 3D tissue constructs. In this work, we leverage a subtractive manufacturing technique known as laser-based hydrogel degradation (LBHD) to guide collective epithelial morphology and exert spatiotemporal control over cell differentiation. We demonstrate the ability to carve features at high resolution within 3D tissue constructs. This thesis demonstrates the potential to direct clusters of salivary cells to migrate through the tunnels carved into hydrogels, form open lumens, and obey branching cues to form a rudimentary gland. This work has the potential to contribute to our understanding of how to create microscale glandular tissues.
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    Engineering Hyaluronic Acid Hydrogels to Enable Ex Vivo 3D Culture of Patient Derived Xenografts and Associated Stroma in a Perfusable Microfluidic Platform
    (2022-12-02) Bonteanu, Andrei; Grande-Allen, K. Jane; Harrington, Daniel A
    Despite the recent availability of physiologically relevant in vitro cancer models such as organoids and spheroids, two-dimensional (2D) monolayer culture and animal models remain frequently used in preclinical drug development. Current in vitro models used in industry are compatible with high-throughput screening, yet are unable to recapitulate tumor microenvironment (TME) heterogeneity, a critical need that this thesis aimed to address. The objective of this thesis was to engineer a hyaluronic acid (HA) hydrogel to enable three-dimensional (3D) ex vivo culture of patient derived xenografts (PDX) and associated stroma in a perfusable microfluidic platform amenable to high-throughput screening. To achieve this, I investigated the impact of tailoring hydrogel composition and network structure for cancer-relevant fibroblasts in a perfusable microfluidic drug- screening microplate. The first part of this thesis demonstrated the importance of tuning the hydrogel parameters of crosslink density, bioligand functionalization, and network degradability on fibroblast morphology and viability. This work underscored the importance of formulating a hydrogel that could maintain structural integrity for long-term 3D culture without negatively impacting embedded cells, providing a context for rational hydrogel design within the scope of high-throughput microfluidic screening platforms. After addressing the model extracellular matrix (ECM), I sought to recapitulate stromal encapsulation of prostate cancer cells by building a Core-Shell organization of fibroblasts wrapped by a uniform layer of fibroblasts. The second part of this thesis used this Core-Shell architecture to build a complex four-compartment in vitro metastatic prostate cancer model, consisting of prostate cancer PDXs, bone marrow derived fibroblasts, and monocytes alongside an endothelial microtubule within a perfusable microfluidic drug-screening plate. The representative nature of the model was achieved by the complexity of cell types represented, including heterogeneity inherent to the PDX cancer cell source. The interaction within this in vitro TME can be used to generate insight into how crosstalk between stromal and cancer cell compartments can impact cancer cell survival, a phenomenon that current simple 2D and 3D models do not have the capability to represent. Lastly, this work tested the feasibility of the model for drug screening on a diverse panel of PDXs using clinically relevant anti-cancer drugs.
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    Engineering Vascularized Hepatic Tissue in Bioactive Poly(ethylene glycol)-based Hydrogels
    (2013-09-20) Higbee, Steven; West, Jennifer L.; Grande-Allen, K. Jane; Harrington, Daniel A
    Transport of oxygen and nutrients to cells within engineered tissues remains one of the most significant challenges in tissue engineering. This challenge has led researchers to seek new strategies to engineer vascularized tissues. Co-cultures of endothelial cells and pericytes can be used to form microvascular networks in bioactive scaffolds, and these networks have been shown to be perfusable and capable of anastomosis with host vasculature. These co-cultures are prevalent in the literature; however, little investigation has been done into the combination of cell-formed microvasculature with parenchymal cells. In our work, we used a co-culture approach to grow microvascular networks in a biomimetic poly(ethylene glycol) (PEG) hydrogel, in the presence of functional hepatocytes. Through the simultaneous encapsulation of three cell types – endothelial cells, pericyte precursors, and hepatocytes – in our biomimetic PEG system, we successfully engineered vascularized hepatic tissue. These vascularized tissues exhibited two distinct benefits when compared to non-vascularized controls. First, incorporation of the vasculogenic cells led to significant improvements in hallmark hepatocyte functions. Hepatocytes encapsulated alongside the vasculogenic cells demonstrated improved albumin synthesis and cytochrome P450 enzyme activity. These improvements result from physical and chemical cues from non-parenchymal cells, which regulate hepatocyte function in vivo and in vitro. Second, the cell-formed microvasculature led to improved mass transport within the hydrogel. In a microfluidic culture system designed to investigate the functionality of the cell-formed microvasculature, we demonstrated that the cell-formed networks are capable of anastomosis with prefabricated channels within the device. Further, transport through these networks significantly increased the distance from a media channel over which hepatocyte viability was supported. Our results suggest that a combination of prefabricated conduits and cell-formed microvasculature may be influential in the scaling up of engineered tissues.
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    Role of cell-extracellular matrix interactions on tissue morphology, cell phenotype and organization in a salivary gland regeneration model
    (2019-04-17) Martinez, Mariane; Harrington, Daniel A
    Head and neck cancer affects over 64,000 Americans every year. Most of these patients receive radiation therapy as part of their standard treatment, which leads to irradiation-induced xerostomia (dry mouth). Xerostomia drastically impairs these patients’ quality of life. Our lab proposes to develop a functional gland, suitable for re-implantation, from salivary-derived human stem/progenitor cells (hS/PCs) isolated from the patient before radiation therapy. Biocompatible hyaluronic acid (HA)-based hydrogels, functionalized with extracellular matrix (ECM)-derived peptides and seeded with hS/PCs and other support cells, will be used as a scaffold for bioengineering an autologous salivary gland replacement. The first part of this work showed that hydrogel porosity and peptide content impacted hS/PC viability and 3D organization. Peptide-modified hydrogels maintained high hS/PC viability and yielded larger multicellular structures that better resembled a developing salivary gland. Use of an integrin ligand led to a higher number of multicellular structures and enhanced hS/PC proliferation. Specifically, migration-permissive (MP-HA) hydrogels led to the highest activation of integrin β1. In short, these experiments defined a hydrogel parameter space for hS/PC encapsulation and 3D culture that avoids confined, spheroidal multicellular assemblies in favor of asymmetric structures with early peripheral buds. Such features bring the models closer to the observed behavior of branching epithelial buds during salivary morphogenesis. The second part of this work describes the isolation and characterization of human salivary-derived fibroblasts (hSFs), and their implementation in co-culture with hS/PCs. hSF-hS/PC 3D encapsulations were conducted either as fully mixed co-cultures, or as adjacent but discrete bilayers. Mixed co-cultures led to significantly higher overall cell viability and structure formation than discrete bilayer co-cultures and monocultures of either cell types. hSFs expressed basement membrane proteins in mixed co-cultures; basement membrane accumulated most in between single hSFs and multicellular hS/PC structures. Lastly, time-lapse imaging of mixed co-cultures illustrated that single cells and multicellular structures composed of either or both cell types were dynamically migrating and reorganizing in MP-HA over time. Thus, the use of MP-HA and a mesenchymal cell type enhanced overall cell viability, growth, and organization in a salivary gland regeneration model.