Abstract
Stem cells are integral to the development of regenerative therapies and disease models. They are characterized by their unique ability to self-renew and differentiate into all cell lineages including those with limited ability to either regenerate or expand (e.g., neural and cardiac cells). In in vivo development of embryonic stem cells, this differentiation is driven by environmental signals. Due to their complexity and level of detail, controlling the fate of embryonic stem cells through the 3D microenvironment has been technologically out of reach to date. However, the continuous and accelerating development in additive manufacturing and biopolymer synthesis might have found the answers to achieving such a goal. In this thesis, fine-tuning and guiding stem cell differentiation is achieved by incorporating additive manufacturing into mainstream developmental biological sciences. 3D printing enable the fabrication of confinements, which, through their geometry, control the cell number, packing density, geometry, shape, and size of the 3D stem cell aggregates, known as embryoid bodies (EBs). These EBs, which aim to replicate a native embryo’s structure, are a promising tool for mimicking embryonic development and controlling differentiation into cell lineages. However, unlike adult cells, embryonic development is a self-sustained process based on intercell interactions within a supportive environment that isolates the system from exogenous interactions (e.g., oviparous eggs). Therefore, the development and maturation of the EBs required the development of novel micro-environments that reproduce those conditions. The free radical oxidation of cellulose (derived from plants), a structural,sustainable and biocompatible biopolymer that is exogenous to mammals, enabled the fabrication of hydrogels suitable for supporting the growth of EBs while preventing their dispersion. The anionic surface of the hydrogel provides an enviroment in which culture stem cells can develop for an extended period without compromising the encapsulated cells’ pluripotency. Given that mammalian cells lack the tools to synthesize or degrade cellulose, an enzyme extracted from Trichoderma reesei was used to degrade the matrix and release the intact cells selectively. To optimize the process and the quality of the results, we also explored the biosynthesis and extraction of a purer version of cellulose from bacterial sources. Despite the different processing and extraction methods, the final cellulose hydrogels derived from both sources (i.e., plant and bacteria) did not show remarkable differences when used as bio-inks to print a cell-laden 3D scaffold. In all cases, the results were free-standing hydrogels with adequate mechanical characteristics, which eliminated the need for chemical stabilizers and other extrinsic components for their fabrication. The interactions of the support matrix with the EBs were controlled through the surface charges. By contrast, the interaction with the exterior (e.g., degradation rate and nutrient diffusion) was controlled with the scaffold’s geometry. Variation of the geometry, which was used to control the constructions’ surface/volume ratios, enabled optimization of the degradation rate and quick release of the encapsulated cells. This finding broadened the application of the bio-ink beyond organ printing and into the field of cell therapies, where they could be used for the transport and delivery of cells at a predefined site and rate. This thesis establishes digital manufacturing and material science principles to set a foundation for technology to communicate and interact with populations of stem cells through geometrical and mechanical cues. The results demonstrate a new level of control in stem cell biology derived from its convergence with the rapidly developing fields of additive manufacturing and biomaterials science.