Engineering Microporous Annealed Particle Scaffolds as a 3D Stem Cell Culture Platform Through the Presentation and Preservation of Native Bioactive Signaling Cues

Design of biomaterial scaffolds for stem cell expansion can be improved by mimicking a cell’s native 3D microenvironment while minimizing cellular stressors that may bias cell fate or reduce functionality. This is particularly important when manufacturing stem cells for clinical therapies, which requires extended in vitro culture to achieve therapeutically relevant cell doses due to the increased risk of dynamic changes in stem cell phenotype and potency. Hydrogel biomaterials can be designed to provide sophisticated mimics of the cellular microenvironment but have yet to show commercial success for stem cell manufacturing. Synthetic hydrogel systems are traditionally supplemented with bioactivity through covalent immobilization (e.g., adhesion proteins or moieties) or exogenous addition of signaling cues (e.g., growth factors). These approaches are often inefficient as they do not account for changes in the conformation and effectiveness of native proteins or fail to replicate the complexity of signaling interactions in vivo. Furthermore, the common use of digestive enzymes to subculture cells or harvest them from synthetic hydrogel platforms causes cellular stress and destroys the beneficial cell-secreted ECM that serves to replicate native signaling. As the clinical use of cell-based therapies becomes more popular, hydrogel technologies that address these challenges could enhance the potency of manufactured cells.

Microporous annealed particle (MAP) scaffolds are a relatively new class of biomaterial composed of an injectable slurry of hydrogel microspheres which undergo a secondary crosslinking mechanism to form a solid scaffold with cell-scale porosity. The building-block design of MAP scaffolds is inherently scalable, and the interconnected porosity enables unrestricted diffusivity which creates a more homogenous culture environment than traditional nanoporous hydrogels. These properties make MAP scaffolds an intriguing candidate for the large-scale expansion of stem cells.

In this dissertation, we present enhancements to MAP scaffolds as a 3D culture platform in the areas of bioactivity and cytocompatible harvesting. Glycosaminoglycans (GAGs) are natural polysaccharides commonly used in biomaterials that non-covalently bind growth factors to enhance their stability and sequester them to the cell surface. We incorporate GAGs into MAP scaffolds by developing methods that allow them to more closely mimic their native presentation. We also design and develop a photodegradable MAP scaffold to provide a non-enzymatic cell harvesting mechanism for reducing cellular stress and preserving cell secreted ECM. Overall, we believe the work in this dissertation advances MAP scaffolds as a synthetic microenvironment and furthers its potential as a scalable platform for the manufacture of clinically relevant cell types.