Blood vessel mimics (BVMs) are tissue engineered blood vessels that are intended as an intermediate testing environment for intravascular devices, such as stents. Specifically, Cal Poly’s Tissue Engineering Lab hypothesizes that BVMs can be used to test endothelial cell and smooth muscle cell responses to existing and new vascular stents. Characterization techniques are required for BVMs to be accepted as a valid testing model, prior to being employed as an in vitro model to determine the effects of medical treatments. Quantitative real-time polymerase chain reaction (qPCR) is one available option for evaluating gene expression of tissues. qPCR can be performed on DNA synthesized from RNA isolated from cells, and in this application, will provide quantitative information on what proteins where being transcribed within the cells at the time of RNA isolation. qPCR can be used to determine the proteins expressed in BVMs at baseline in order to then characterize changes in protein expression induced by stent deployment within the BVM. The aim of this thesis was to optimize existing qPCR protocols, and implement the optimized protocols to characterize gene expression of stented and unstented blood vessel mimics (BVMs) and cells from a donor with Diabetes grown in Cal Poly’s Tissue Engineering Laboratory. To accomplish this goal, existing qPCR protocols were evaluated and modified to ensure reproducible, valid results were produced. Standard operating procedures were created for RNA isolation, cDNA synthesis, qPCR and qPCR data analysis. Optimized qPCR methods were then applied to BVMs from umbilical and coronary cell sources to compare the models and to study the BVM responses to stent deployment. Additional primers were also identified for potential usage as reference genes and as diabetic markers for diseased BVMs.

Blood vessel mimics (BVMs) are simple tissue engineered blood vessel constructs intended for preclinical testing of vascular devices. This thesis developed and implemented methods to characterize two of these components. The first aim of this thesis investigated the effect of cell culture duration and flow conditions on endothelial cell gene expression, especially regarding endothelial-to-mesenchymal transition (EndMT). A trend of decreased endothelial marker gene expression and increased mesenchymal marker gene expression would indicate EndMT. qPCR analysis revealed that increased cell culture duration did not result in EndMT, and in fact increased endothelial marker expression as cell culture duration increased. Disturbed flow conditions decreased endothelial marker and increased mesenchymal marker expression relative to static culture. The second aim of this thesis developed methods to determine cytotoxicity of, and endothelial cell adhesion to, novel BTEAC salt scaffolds. Immunostaining was used to visualize these scaffold effects. The cytotoxicity elution assay showed that BTEAC salt scaffolds were not more cytotoxic than the standard PLGA scaffold. Direct contact assays spanning several timepoints also found that BTEAC salt scaffolds were not more cytotoxic than standard scaffolds but had higher endothelial cell adhesion and coverage than standard scaffolds. Overall, this thesis developed and implemented methods to characterize the endothelial cells used in the BVM model.