ENGINEERING THREE-DIMENSIONAL MODELS OF MICROTISSUE DEVELOPMENT

This research addresses the need for more accurate three-dimensional, biomimetic, in vitro models to replicate and study in vivo processes outside normal physiological contexts. Three models are explored in this work: anisotropic 3D confinement, vasculogenesis in decellularized matrix, and curved anisotropic tubular hydrogels. The aim was to improve in vitro tools used to study tissue development and accelerate development of functional tissue grafts, an issue of paramount importance in biomedical engineering. First, the role of anisotropy was studied, as it is important in scaffold design and tissue functionality. Anisotropic 3D confinement was found to affect the growth and development of human fetal lung fibroblasts (WI-38) in microfabricated 3D wells. WI-38 increasingly aligned along the long axis of rectangular wells with increasing aspect ratios. Interestingly, aligned cells propagated these external confinement cues to the natural matrix microenvironment, depositing fibronectin in an anisotropic pattern. To quantify cellular, nuclear, and matrix anisotropy, a robust automated autocorrelation analysis was developed using a new value-weighted autocorrelation approach, which represented a more dynamic range of data than the usual unweighted method. Actin, microtubules, and Rho-associated coiled-coil kinase (ROCK)-mediated actomyosin contraction were found to be key modulators of shape-specific anisotropic effects. Next, the effectiveness of the WI-38 matrix microenvironment to stimulate EC tube formation was explored, given the matrix’s importance in sensing and communicating external cues. A 3D model for human, tissue-specific vasculogenesis was developed using decellularized WI-38 matrix and human pulmonary microvascular endothelial cells. Vascular networks formed successfully in vitro, displaying positive vessel-like properties, and also increased in density under hypoxic conditions. Lastly, to approach physiological geometry, a 3D mimetic hydrogel model was developed. Self-assembling, bilayer hydrogels were created in the form of curved, anisotropic tubes, inspired by ductal and acinar geometries in mammary tissue. Photoencapsulated MDA-MB-231 cells were viable in PEGDA bilayer hydrogel tubes (15 days). SUM159 cells adhered to the surface of and proliferated (9 days) on bilayer hydrogels tubes made of a novel methacrylated gelatin PEGDA mixture. In summary, this research improves our understanding of the 3D microenvironment by bringing together cell biology, materials science, and engineering to better mimic physiological processes