Vascularizing Tissues Ex Vivo: Engineering Microvascular Structures and Cellular Phenotypes

Numerous approaches to vascularizing engineered tissues ex vivo have been explored for relatively low-volume constructs; however, the development of the methods and biological understanding necessary to engineer a range of construct dimensions with physiologically-accurate microvasculature remains ongoing. This thesis focuses on several aspects of this challenge, including (i) the fabrication of vascular channels using 3D bioprinting, (ii) assessment of collagen, fibrin, and collagen-fibrin co-gels as scaffold materials, and finally (iii) exploring the suitability of iPSC-derived endothelial cells (ECs) for engineering vasculature. In the case of (i), a bioprinter was designed and sacrificial materials assessed for characteristics relevant to their use in patterning vascular channels within hydrogels (e.g., biocompatibility, mechanical properties). The most promising sacrificial materials, Pluronic F-127 (35% w/v) and gelatin-hyaluronan (5.25%, 3% w/v), had sufficient viscosity to create precisely patterned channels and exhibited no cytotoxicity. With regard to (ii), it was found that fibrin and co-gels facilitated EC sprouting, whereas collagen elicited migration of individual ECs, and that PKC-epsilon phosphorylation may play a role in regulating these differential responses. Overall, co-gels were found to be superior to either hydrogel alone as fibrin both enabled EC sprouting and impeded cell-mediated contraction, while increasing collagen within co-gels resulted in a progressively higher compressive modulus and enabled the formation of microchannels that could be perfused up to ~15 mmHg when composed of 2.5 mg mL-1 fibrin and 3 mg mL-1 collagen. In terms of (iii), RNA-seq was performed on iPSC-ECs and primary ECs (HUVEC, HIAEC, and HdMVEC) and revealed that iPSC-ECs have upregulated genes related to arterial hemogenic endothelium, indicative of possible immaturity. Additionally, differential expression of inflammation, hemostasis, shear-sensing and barrier function-related genes was discerned. Favourably, iPSC-ECs had an intermediate angiogenic capacity compared to native ECs. Overall, iPSC-ECs displayed promising similarities to native ECs, however, several key aspects of their function were identified as requiring further evaluation and improved maturation. Ultimately, this thesis has contributed to the advancement of biomaterials and the cell sourcing necessary for developing vascularized tissues and their concomitant clinical and scientific applications.Ph.D.2021-07-01 00:00:0