Graduate College

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The doctoral dissertation and master's thesis reflects the scholarly research in a graduate program as required for the completion of the degree at the University of Delaware.

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Browsing Graduate College by Subject "3D cell culture"

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    Design of synthetic extracellular matrices for 3D culture models for the study of disease
    (University of Delaware, 2018) Ovadia, Elisa
    Improved approaches for culturing mammalian cells in vitro are needed to study cell response to environmental cues and better understand disease progression. Three-dimensional (3D) biomaterials are a powerful tool for culture of cells in a physiologically relevant system. In tissues, cells reside in a niche or extracellular matrix (ECM), where key structural proteins (i.e., collagen, laminin, fibronectin) and mechanical properties influence cell behavior. Specifically, synthetic biomaterials afford control for tuning of mechanical and biochemical properties to mimic in vivo niches. In this thesis work, we have developed new tools for modulating both mechanical and biochemical stimuli, established a three-dimensional stem cell culture platform, and developed a three-dimensional breast cancer dormancy model. ☐ Initially, we examined tuning of mechanical and biochemical properties of poly(ethylene glycol) (PEG) hydrogels by development of new tools. Initial mechanical tuning of PEG hydrogels was accomplished by use of 455 nm visible light-initiated thiol-ene ‘click’ chemistry, where irradiation time and light intensity were used to control mechanical properties. Rheological measurements showed decreased mechanical properties of visible light-formed hydrogels compared to UV initiated hydrogels. Magic-angle spinning confirmed slower gelation rate kinetics suggesting defects contribute to mechanical properties differences. The ‘free’ defect end groups were then utilized by reacting and stiffening the network at a later time. ☐ Tuning of hydrogel biochemical properties such as whole protein presentation and peptides with secondary conformation was accomplished by use of orthogonal handles. Proteins are important in regulating cell growth, proliferation, and differentiation. Here, we utilized strain promoted azide-alkyne cycloaddition (SPAAC) for facile conjugation of a model fluorescent protein that was site-specifically modified with i) an azide handle for hydrogel incorporation and ii) an enzymatic cleavable site. Ultimately, we demonstrate the orthogonality of these functionalities by formation of a three-layered protein-incorporated hydrogel, and later removal one of the proteins, while the hydrogel remained intact. Confocal imaging was used to confirm fluorescent protein incorporation, layering, and removal. This demonstrated a versatile on-demand approach for spatiotemporal presentation of proteins utilizing bio-orthogonal methods. Additional use of orthogonal modalities was examined by sequential orthogonal click reactions, specifically photoinitiated thiol-ene and SPAAC for peptide cyclization and conjugation. This approach for the design of functionalized cyclic peptides was established for use in 3D cell culture biomaterials and in cell targeting biomedical applications. ☐ Induced pluripotent stem cells (iPSCs) are of interest for the study of disease, where these cells are patient-derived and can differentiated into any cell types; however, 3D culture and differentiation of iPSCs for these applications remains limited. Here, we used synthetic hydrogels that allow precise presentation of specific biochemical cues for 3D culture to test the hypothesis that iPSC viability could be rescued with appropriate biochemical cues inspired by proteins and integrins important for iPSC culture on Matrigel. Motifs inspired by iPSC binding to Matrigel, including laminin-mimics IKVAV and YIGSR, a5b1-binding PHSRNG10RGDS, avb5-binding KKQRFRHRNRKG, and RGDS were selected. YIGSR and PHSRNG10RGDS had the highest iPSC viability, where binding of 1 integrin was key, and these permissive compositions also allowed iPSC differentiation into neural progenitor cells. In sum, we established synthetic matrices for the encapsulation, culture, and differentiation of iPSCs for studies of cell-matrix interactions and deployment in disease models. ☐ Lastly, in the body, disseminated breast cancer tumor cells migrate to a metastatic site where they can remain dormant or not-proliferating for years, such as from estrogen-receptor positive (ER+) cells, before re-activating. Interactions with the extracellular matrix surrounding these cells, is hypothesized to play a role in the dormancy to reactivation cascade; however, knowledge of how matrix properties influence cancer cell behavior is limited. To address this, we developed a synthetic 3D hydrogel in vitro model to probe effects of mechanical and biochemical properties on regulating breast cancer cells. Formation of ER+ dormant micrometastases was detected after 40 days in culture and quiescent cell behavior was observed. Bioinformatic tools were applied to evaluate cell response and dormancy in cultures. Overall, we have established a robust model for systematically evaluating breast cancer cell-ECM interactions for applications in improving methods for cancer detection and identifying potential therapies. ☐ Collectively, the research described here highlights advances in tools for modulation of properties of synthetic PEG 3D cultures. Application of these 3D biomaterials was used for establishment of i) three-dimensional culture and differentiation of iPSCs and ii) a breast cancer disease model.
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    Directing stem cell behavior in an engineered vocal fold-like microenvironment
    (University of Delaware, 2019) Zerdoum, Aidan
    The overall goal of this doctoral dissertation is to understand how microenvironmental cues direct the phenotype and function of human mesenchymal stem cells (hMSCs) in the context of vocal fold (VF) tissue repair and regeneration. To assess the therapeutic efficacy of stem cell-based therapies for the treatment of vocal fold scarring, VF-mimetic hydrogels were developed consisting of a covalent hyaluronic acid (HA)-based network interpenetrated by assembled collagen fibrils. When hMSCs were cultured in these gels, they adopted a phenotype and expression pattern reminiscent of cells involved in the wound healing process. To understand the effects of phonation on hMSC differentiation, a bioreactor capable of the induction of high frequency (200 Hz) oscillatory air pressure was designed, constructed and characterized. Vibratory culture of hMSCs in HA/collagen gels over a 3-day, 1h-on 1h-off cycle resulted in significant downregulation of fibrogenic markers and loss of alpha smooth muscle actin positive staining, indicating attenuation of myofibrogenic potential and a further shift away from a fibrotic phenotype. Finally, the roles of connective tissue growth factor (CTGF) in promoting tissue fibrosis were investigated. hMSCs were encapsulated within a cell-adhesive, protease-degradable HA network devoid of any collagen fibrils and cell culture was maintained in CTGF-conditioned media for 21 days. CTGF promoted fibrogenesis through mitogen activated protein kinase (MAPK)-dependent regulation. Overall, this work presents several in vitro models for future therapeutic endeavors aimed towards limiting scar formation in VF tissues.
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    Stereoselective synthesis of butenolides and its applications in total synthesis of sessilifoliamides and development of new chemical tools for hydrogel based biomaterials
    (University of Delaware, 2017) LI, YI
    The research in my doctoral study consists of two major parts. One part is a synthetic method development and its applications in solving complex synthetic chemistry problems. The other part is a multidisciplinary research involving the collaboration of the Fox group and the Jia group in material science, studying the development of new biomaterials for cell culture purpose. ☐ Butenolides are privileged scaffolds in medicinal chemistry and important synthetic building blocks. Allyl cyclopropene carboxylates undergo ring expansion reaction to give alloxyfuran intermediates, which can further rearrange to Δβ, γ butenolides via Claisen rearrangement or Δα, β butenolides via a further Cope rearrangement. The chemoselectivity and regioselectivity can be controlled through judicious choice of catalyst and reaction temperature. The transformation is stereospecific and transfers the chirality of the allyl cyclopropene carboxylate to the butenolide product. Through assignment of the absolute configuration of the product, it was possible to propose a mechanistic model for the rearrangements. ☐ The second chapter centers on the application of the butenolide formation method described in chapter one towards the total synthesis of sessilifoliamide nature products. The route features a one-step installation of the butenolide moiety to furnish the dehydro-sessilifoliamide from a cyclopropene carboxylate precursor and a short and efficient synthesis of the cyclopropene carboxylate. A short 11-step synthesis of dehydro sessilifoliamide B is described. Attempts to prepare sessilifoliamide B by hydrogenation were unsuccessful. ☐ The third chapter describes a collaborative research on the development of biomaterial for cell culture purpose using tetrazine ligation. The chapter describes the development of chemical tools for the hydrogel preparation. The mechanical properties of hydrogels were controlled by introducing capping groups alongside crosslinker molecules in interfacial crosslinking events. Enzyme degradable crosslinkers and cell adhesive signals are prepared and introduced to the gels. It was shown by my collaborator that all the newly developed chemical tools can be incorporated in to the HA-Tz based hydrogels and NIH 3T3 cells can be cultured in the system with good viability and morphology.