Cardiovascular disease (CVD) is the leading cause of death internationally. Efforts to decrease CVD death has been explored through stem cell technology, specifically organoid formation. Current cardiac organoid models lack the vascular networks for nutrient supply and maturation. In this study, pillar perfusion technology is used to fabricate cardiac organoids and induce vascularization via dynamic culturing and the addition of vascular promoting growth factors (GFs). In addition to this study, a millifluidic chip is engineered for shear stress application via flow simulations and experimental flow analysis. We successfully optimized the millifluidic chip to achieve fluid shear stress of 20mPa and validated through particle tracking velocimetry using 0.1um diameter beads under flow. The results of cardiac organoids displayed contraction and growth of endothelial cells (ECs) under dynamic flow with GFs. In addition, smooth muscle cells (SMCs) displayed growth via GFs in both dynamic and static culturing. Although vascular networks were not present in all conditions of this experiment, this thesis can serve a basis for searching other methods of inducing vascularization.

Electrospun nanofibers have been researched extensively in the culturing of stem cells to understand their behavior since electrospun fibers mimic the native extracellular matrix (ECM) in many types of mammalian tissues. Here, electrospun nanofibers with defined structure (orientation/alignment) and pore size could significantly modulate human mesenchymal stem cell (hMSC) behavior. Controlling the fiber membrane pore size was predominantly influenced by the duration of electrospinning, while the alignment of the fiber membrane was determined by parallel electrode collector design. Electric field simulation data provided information on the electrostatic interactions in this electrospinning apparatus.hMSCs on small-sized pores (~3-10 µm²) tended to promote the cytoplasmic retention of Yes-associated protein (YAP), while larger pores (~30-45 µm²) promoted the nuclear activation of YAP. hMSCs also displayed architecture-mediated behavior, as the cells aligned along with the fiber membranes orientation. Additionally, fiber membranes affected nuclear size and shape, indicating changes in cytoskeletal tension, which coincided with YAP activity. The mechanistic understanding of hMSC behavior on defined nanofiber structures seeks to advance their translation into more clinical settings and increase biomanufacturing efficiencies.

Metastatic cancer is more dangerous and difficult to treat than pre-metastatic cancer. Ninety percent of cancer-related deaths are caused by metastatic cancer. When cells go through metastases, they go through changes that allow them to break away from the primary tumor and invade secondary tissues. These changes, in lipid membrane composition and cellular glycocalyx, make the cell more resistant to therapeutics. Actin cytoskeleton contractility plays a major role in these changes, as increased contractility has been linked to upregulation of phosphoinositides and production of glycoproteins. Light induced molecular adsorption of proteins (LIMAP) was used to control the actin arrangement and cell shape in order to mimic and study metastatic cells. Negatively charged proteins electrostatically adhere to the surface in order to create patterns for the cells to stick. Neutravidin was conjugated to poly(glutamic acid) to improve attachment to the surface. We observed differences in cell shape and phosphoinositide behavior based on LIMAP patterning. Additionally, expression of key glycoproteins related to cancer metastasis increased with increased actin contractility. The actin cytoskeleton was the main driver of changes to the cell membrane and glycocalyx.

Familial hypertrophic cardiomyopathy (HCM) is a genetic disease largely caused by a mutation in myosin binding protein C (MYBPC3) and it affects about 1:500 population leading to arrhythmic sudden death, heart failure, and atrial fibrillation. MYBPC3 activates calcium-induced actin-myosin filament sliding within the cardiac sarcomere, creating the force necessary for heart contraction. The underlying molecular mechanisms causing HCM phenotype remain elusive, therefore, there is an urgent need for a reliable in vitro human HCM model to investigate the pathogenesis of HCM. This study utilized isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with MYBPC3 gene mutation (wildtype, heterozygous, homozygous) and further micropatterned them into fiber-like structures on polyacrylamide hydrogels of physiological and fibrotic-like stiffnesses. Cells were cultured for an extended culture time up to 60 days and their morphology/attachment, contractility, and calcium transient were extensively and carefully evaluated. It was found that MYBPC3 knockout cells maintained the highest contraction amplitude, but had increased contraction, and relaxation durations, decreased calcium transient amplitude, as well as time to peak and decay times over the culture period in comparison to the isogenic wildtype. Overall, this study demonstrates that hiPSC-CMs can be successfully patterned and cultured for an extended time on hydrogels forming end-to-end …

This study focuses on engineering whole cell-based biosensors for heavy metal detection. Through the exploitation of metalloregulatory proteins, fabrication of metal ion-responsive biosensors is achieved. Metalloregulatory proteins of the SmtB/ArsR family including arsenite-responsive ArsR, cadmium-responsive CadC, zinc-responsive CzrA, and nickel-responsive NmtR were evaluated as biosensor sensing modules. Characterization of these four metal sensing modules was accomplished through quantification of a reporter green fluorescence protein (gfp) gene. As such, biosensors pCTYC-r34ArsR-pL(ArsOvN)GFP and pCTYC-r34CadC-pL(CadOv1)GFP displayed excellent gfp expression and sensitivity to As(III) and Cd (II), respectively. These two biosensors were consequently selected and successfully implemented in soil bacterium Pseudomonas putida. Lastly, a proof of concept arsenite-responsive genetic toggle switch is proposed utilizing PurRcelR467 (PC47), a cellobiose-responsive gene, and an LAA degradation tag. Overall, this study expands the bank of metalloregulatory bioparts for heavy metal sensing in the aim of constructing an optimized water monitoring system.