Design of synthetic extracellular matrices for 3D culture models for the study of disease
| Author(s) | Ovadia, Elisa | |
| Date Accessioned | 2022-09-22T12:04:24Z | |
| Date Available | 2022-09-22T12:04:24Z | |
| Publication Date | 2018 | |
| SWORD Update | 2022-08-11T19:09:37Z | |
| Abstract | 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. | en_US |
| Advisor | Kloxin, April M. | |
| Degree | Ph.D. | |
| Department | University of Delaware, Department of Chemical and Biomolecular Engineering | |
| DOI | https://doi.org/10.58088/vgq1-0955 | |
| Unique Identifier | 1345467595 | |
| URL | https://udspace.udel.edu/handle/19716/31400 | |
| Language | en | |
| Publisher | University of Delaware | en_US |
| URI | https://login.udel.idm.oclc.org/login?url=https://www.proquest.com/dissertations-theses/design-synthetic-extracellular-matrices-3d/docview/2708570037/se-2?accountid=10457 | |
| Keywords | 3D cell culture | |
| Keywords | Biomaterials | |
| Keywords | Breast cancer recurrence | |
| Keywords | Disease model | |
| Keywords | Extracellular matrix | |
| Keywords | Induced pluripotent stem cells | |
| Title | Design of synthetic extracellular matrices for 3D culture models for the study of disease | en_US |
| Type | Thesis | en_US |