Development of 3D Human Scaffold-free Functional Vascular Tissue Models Using Cellular Self-assembly

DeMaria, William

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Development of 3D Human Scaffold-free Functional Vascular Tissue Models Using Cellular Self-assembly

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Cardiovascular disease is the leading cause of death in the United States, accounting for ~700,000 deaths and $219 billion of health care expenses annually. Currently, cardiovascular research heavily relies on two-dimensional (2D) cell culture and animal models, which often fail to predict the clinical success of a new drug. To increase the predictive capabilities of pre-clinical trials, researchers have developed in vitro models that utilize human cell types. However, these models often lack the complex three-dimensional (3D) microenvironment in which the cells reside, do not introduce physiological shear stresses due to flow, and do not utilize human vascular cell types such as smooth muscle cells (SMCs) and endothelial cells (ECs). Tissue engineering has shown potential for generating more complex 3D vascular tissue models that consist of functional human SMCs and ECs in a tubular construct (i.e., tissue engineered blood vessel (TEBV)), but most approaches rely on the use of a scaffold and do not co-culture SMCs and ECs in 3D. These limitations prevent the proper cell-cell and cell-extracellular matrix (ECM) interactions necessary to create a fully differentiated and functional vascular model. In contrast, scaffold-free TEBVs (i.e., TEBVs that consist of cells and cell-derived ECM only) exhibit greater cell density, enhanced ECM production, and improved biological function, all of which are important to recapitulate native vascular structure and function. Thus, the overall goal of this dissertation was to create a scaffold-free TEBV from human cells that responds to vascular agonists and withstands physiological shear stresses. To do so, human mesenchymal stem cells (hMSCs) and ECs were co-seeded into non-adhesive agarose wells to create hMSC-EC tissue rings. hMSCs were chosen as the cell source because they can be differentiated into SMCs and exhibit an increased proliferative capacity compared to somatic SMCs. Varying concentrations of ECs (0, 10, 20, and 30% ECs) were co-seeded with hMSCs to investigate the effects of ECs on hMSC-derived SMC differentiation and tissue ring contractility in response to endothelin-1 (ET-1), a potent vasoconstrictor. Differentiation was evaluated using immunostaining and western blotting for SMC contractile proteins, while contraction was measured using wire myography. hMSC-EC rings (20 and 30% ECs) exhibited significantly enhanced contractile protein expression (transgelin, calponin, and smoothelin) as well as ET-1 mediated contraction compared to the 0% EC rings. hMSC-EC rings were then stacked and fused together to form tubular hMSC-EC TEBVs. Mechanical stimulation via cyclic circumferential distension was then used to strengthen the TEBV constructs and achieve physiological shear stresses within TEBV lumens. The effects of three mechanical stimulation regimens with varying cyclic distension magnitudes and culture times on hMSC-EC TEBV morphology and hMSC-SMC differentiation were evaluated. The optimal regimen, 22 days of static culture (i.e., unstimulated), resulted in TEBVs that exhibited aligned cells, expressed contractile proteins and mature collagen fibers, and withstood ~3 dyne/cm2 shear stress and ~20 mmHg pressure for six days of luminal flow and pressure. In summary, this work demonstrated a scaffold-free approach for creating 3D vascular tissue constructs that mimic native vascular structure and function. In the future, these engineered tissue constructs may be used to bridge the gap between pre-clinical studies and human trials for drug discovery applications.

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