Engineered Vascular Tissue Generated by Cellular Self-Assembly

Gwyther, Tracy A

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Engineered Vascular Tissue Generated by Cellular Self-Assembly

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Small diameter vascular grafts comprised entirely from cells and cell-derived extracellular matrix (ECM) have shown promise in clinical trials and may have potential advantages as in vitro vascular tissue models. A challenge with current cell-derived tissue engineering approaches is the length of time required to generate strong, robust tissue. There is a lack of alternative methods to rapidly assemble cells into a 3D format without the support of a scaffold. Toward the goal of engineering a new approach to rapidly synthesizing vascular tissue constructs entirely from cells, we have developed and characterized a strategy for creating cell-derived tissue rings by cellular self-assembly. The focus of this thesis was to develop the system to rapidly generate engineered tissue rings, and to evaluate their structural and functional properties. To generate tissue rings, rat smooth muscle cells (SMCs) were seeded into round-bottomed, ring-shaped agarose wells with varying inner post diameters (2, 4, and 6 mm). Within 24 hours of seeding, cells aggregated, contracted, and formed robust tissue that could be removed from their wells and handled. If kept in culture, the thickness of these tissue rings increased with time. Mechanical analysis of the tissue showed that it was stronger after only 8 days in culture than engineered tissues generated by other approaches (such as seeding cells in biopolymer gels) cultured and tested at similar time points. Histological staining of the tissue rings revealed high cell densities throughout, along with the presence of glycosaminoglycans and some collagen. We also found that we could use the tissue rings as building blocks to generate larger tubular structures. Briefly, tissue rings were removed from the agarose wells and transferred onto silicone tubing mandrels. Once the rings were placed in contact with each other on the mandrel, they were cultured to allow the rings to fuse together. We found that the ability of tissue rings to fuse decreased with increasing ring “pre-culture” duration, and that we were able to generate fully fused tissue tubes in as little as 8 days (with only one day of ring pre-culture and seven days of fusion). In the last section of this thesis, we established the feasibility of using primary human SMCs to generate self-assembled tissue rings, similar to the self-assembled rings generated with rat SMCs. Compared to the rat SMC rings, human SMC rings were stronger, stiffer and appeared to contain increased levels of collagen. These data showed that human SMCs are capable of self-assembling into tissue rings similar to rat SMCs, and may therefore be used to create engineered human vascular tissue. Overall, we have developed a platform technology that can be used to screen the effects of culture parameters on the structure, mechanics, and function of vascular tissue. We anticipate that through the use of this technology, we can further improve vascular grafts by better understanding factors which promote ECM synthesis and SMC contraction. We can use these results directly toward the generation of vascular grafts by fusing self-assembled cell rings together to form tissue tubes. These novel bioengineered vascular tissues may also serve as a method to produce in vitro models to help further our understanding of vascular diseases, as well as facilitate pre-clinical screening of vascular tissue responses to pharmacologic therapies.

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