Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.Includes bibliographical references (p. 141-153).Creating a physiologically relevant in vitro liver model requires reproducing the cellular heterogeneity of in vivo liver in a functional state. However differentiated sinusoidal endothelial cells (SECs), marked by SE-1 expression are difficult to maintain in culture while stellate cells easily activate and over-proliferate. We hypothesized that recreating a liver tissue system that captured in vivo like paracrine influences would foster survival of these cells, and predicted that stimuli resulting from flow and oxygen gradients close to physiological conditions would preserve the delicate balance between the cell types. Spheroids containing hepatoctyes with incorporated non-parenchymal cells (NPCs) were seeded into capillary bed sized channels in polycarbonate scaffolds, housed in a three-dimensional perfused system, and maintained for two weeks. Micro-flow rates of different media through the formed tissue units in scaffolds were controlled using pneumatic pumps and microfluidics. Staining and confocal imaging of endpoint tissue showed that lower flow rates closer to physiological regimes allowed the survival of SE-1+ SECs, regardless of exogenously added growth factors in the medium. Higher flow rates, exogenous growth factors, and scaffold contact were associated with activation of stellate cells (alpha-smooth muscle actin staining).Since oxygen measurements in the system coupled low flow rates with hypoxic tissue outlet concentrations, we parsed out these variables by repeating flow experiments in low oxygen environments. Retention of SE-i staining cells even in higher flow rates demonstrated that hypoxic conditions in the tissue could play a role in aiding their survival by overcoming negative effects brought about by high flow. The relationship of stellate cells with flow rate was unaffected by oxygen concentrations. To explore if the negative effects of high flow on SE-i expression were mediated by transforming growth factor-beta (TGF-[beta]), we added a TGF-[beta] inhibitor SB-431542 in our cultures, and found that it greatly enhanced the presence of SE-1 staining SECs at high flow rates. In conclusion we successfully created a three-dimensional flow controlled hepatic culture system that allows balanced survival of hepatocytes and non-parenchymal cells, making it useful as a potential model for studies such as cancer metastasis that require interactions between tumor cells and heterotypic host tissue. Key Words: Liver, In vitro, co-culture, sinusoidal, endothelial, stellate, oxygen, flow, shear.by Ajit Dash.Ph.D
