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Title: Vascular network formation via 3D printing and cell-based approaches
Author: Justin, Alexander William
ISNI:       0000 0004 7230 1865
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Vascularization is essential for living tissue and remains a major challenge in the field of tissue engineering. A lack of a perfusable channel network within a large and densely populated tissue engineered construct leads to necrotic core formation, preventing fabrication of functional tissues and organs. While many approaches have been reported for forming vascular networks, including materials processing techniques, such those involving lithography, bioprinting, and sacrificial templating; and cell-based approaches, in which cellular self-organization processes form vessels; all are deficient in their ability to form a vessel system of sufficient complexity for supporting a large cellular construct. What is missing from the literature is a method for forming a fully three-dimensional vascular network over the full range of length-scales found in native vessel systems, which can be used alongside cells and perfused with fluids to support their function. A large number of research groups are thus pursuing novel methods for fabricating vascular systems in order that new tissues and organs can be fabricated in the lab. In this project, a 3D printing-based approach was used to form vascular networks which are hierarchical, three-dimensional, and perfusable. This was performed in thick, cellularized hydrogels similar in composition to native tissue; these being collagen (ECM-like) and fibrin (woundlike), both of which are highly capable of supporting cellular activities, such as cell seeding, cell spreading, and capillary morphogenesis. In order to make use of 3D printed network templates in cellularized hydrogel environments, it was necessary to develop a new approach in which standard 3D printed materials were converted into a gelatin template, via an alginate intermediary, which can be removed quickly in physiologic conditions and which does not reduce cell viability. This multi-casting approach enables a hierarchical channel network to be formed in three-dimensions, capable of being perfused with cell medium to maintain the viability of a cell population, thereby addressing the fundamental problem. Using standard cell staining and immuno-histochemistry techniques, we showed good endothelial cell seeding and the presence of tight junctions between the channel endothelial cells. When fibroblasts were seeded into the bulk of the hydrogel, a high degree of cell viability and cell spreading was observed when a threshold flow rate is met. By counting the number of live and dead cells in a sample regions of the gel, we were able to show a dependency of cell viability upon the perfusion flow rate and further determine a regime in which the vast majority of cells are alive and spreading. This data informs future cellular experiments using this platform technology. The limits of existing 3D printing technology meant that the micro-scale vasculature needed to be formed by other means. Cellular co-culture of endothelial and stromal cell types has been shown to be capable of forming capillary-like structures in vitro. For inclusion with the 3D printed channel system, we investigated the use of an angiogenic method for capillary formation, using multi-cellular spheroids, and a vasculogenic approach, using individual cells, in order that the full vascular system could be constructed. Endothelial and mesenchymal stromal cells were encapsulated in small fibrin and collagen gels and maintained under static culture conditions in order to form capillaries by the above approaches. The aim here was to find a particular gel composition and cell concentration which would support capillary morphogenesis while being suitably robust to handle the mechanical stresses associated with perfusion. As future work, the next step will be to incorporate the vasculogenic co-culture technique, used to form capillary-sized vessels, into a perfusable gel containing the large templated channels, formed via the multi-casting approach. The challenge here is to anastomose the capillary-sized vessels to the large templated channels and thereby enable perfusion of the capillary vessels. This step would be a highly significant development in the field as it would mean large constructs could be fabricated with physiological densities of cells, which could lead to a range of potential therapeutic applications.
Supervisor: Markaki, Athina Sponsor: EPSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Tissue Engineering ; Vascularization ; 3D Printing ; Hydrogel ; Vascular Networks