| Summary: | <p> Three-dimensional (3D) bioprinting is a rapidly growing field that is particularly well suited for “bottom up” tissue engineering, largely due to its ability to control the 3D shape of the engineered construct, as well as its constituents (<i>e.g.</i>, cells and/or material) and their spatial distribution. A variety of nozzle-based techniques have emerged for tissue engineering, and while these excel at building large 3D architectures, they suffer from moderate print resolution and limited printable materials, making them less attractive for smaller, high-resolution constructs. This is due in part to shearing effects and clogging of the nozzle. Thus, alternative printing methods are needed to create smaller constructs requiring high-spatial pattern resolution and size control. </p><p> Our laboratory has previously developed a laser-based biofabrication platform, gelatin-based laser direct-write (LDW) as a technique for bioprinting highly viable cells with spatial resolution unmatched by other printing techniques in 2D. In this thesis, a novel single-step technique was developed to extend this platform to fabricate and spatially pattern 3D alginate microbeads. With this new method, we demonstrate excellent size-control of fabricated microbeads by manipulating the beam diameter used for deposition. We further show that deposited beads have excellent pattern registry, and cells within LDW microbeads maintain high cellular viability. Additionally, we demonstrate that this technique is compatible with our laboratory’s 2D laser direct-writing of cells, illustrating the ability to fabricate spatially-precise, hybrid, 2D/3D cultures of cells and cell-loaded microbeads. Within cellular applications, the mechanical properties of the extracellular matrix have become an important feature for regulating behavior. To further develop our control over the cellular microenvironment, we demonstrate our ability to mechanically tune the stiffness of LDW-printed microbeads, by varying the crosslinking divalent cation and cation concentration used in the LDW microbead fabrication process. Microbead mechanical properties were determined using large printed arrays of microbeads (12 × 12 array) to amplify the resistance generated during traditional compression testing. Using this method, we demonstrated microbead mechanical properties could be tuned by adjusting fabrication and crosslinking parameters, to achieve a wide range of elastic moduli, from physiologic to pathologic values. While this was a valuable step to demonstrate our ability to control aspects of the engineered cellular microenvironment, our alginate structures were still largely limited for cellular interaction due to the lack of adhesion ligands. The inability for cells to interact with the alginate prevents migration within the matrix. </p><p> To overcome the limitations of the inert alginate of our microbeads, we used an established materials processing approach to produce core-shelled microcapsules. This technique consists of coating the printed microbead with a positively charged polymer (<i>e.g.</i>, chitosan or poly-L-lysine), to produce a polyelectrolyte membrane around the bead, then chelating the calcium crosslinking the interior. This resulted in a polymeric shell with an aqueous core entrapping the cellular payload. We found that core-shelled microcapsules from LDW microbeads maintained their pattern fidelity through processing, and encapsulated cells retained high viability. Cancer cells and stem cells encapsulated within these structures were observed to self-assemble to form size-controlled 3D aggregates; tumor spheroids and embryoid bodies, respectively. </p><p> In addition to creating conventional core-shelled microcapsules, we demonstrate that LDW’s spatial precision can be leveraged to produce advanced core-shelled structures of customizable planar geometries, by utilizing single microbeads as voxels, and patterning these in overlapping arrays. Using this technique, we were able to create custom geometries, such as microstrands, bifurcations, rectangular mats, and rings, wherein aggregating cells self-assembled to make continuous three-dimensional aggregates that conform to the shape of the structure. Overall, this doctoral thesis research developed a powerful, laser-based method for engineering custom 3D microenvironments, with applications in tumor modeling and regenerative medicine. These advances hold great promise for fabricating the next generation <i>in vitro</i> diagnostics.</p><p>
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