A defined and automation-compatible macroporous microgel platform for induced pluripotent stem cell-based tissue engineering

In this thesis, a novel technology that utilizes microgels to enable induced pluripotent stem cell (iPSC)-based generation of spheroids in a reproducible, scalable, and high throughput (HTP), automation-compatible manner was developed. The platform enabled the pluripotent expansion and directed differentiation of stem cells within a single three-dimensional (3D) construct, utilizing defined and tunable spherical poly (ethylene) glycol (PEG)-based microgels as a matrix material. The biofunctionalization of the bioinert microgels with vitronectin (VTN) enabled strong cell-material interactions, facilitating the solely cell-guided formation of 3D scaffolds. This structural design without covalent crosslinking allowed for cellular self-organization not only during scaffold formation but also during later developmental reorganization. The incorporation of the microgels into the spheroid further increased the oxygen and nutrient diffusion throughout the construct, enabling large tissue sizes and uniform differentiation. Thereby, this platform was engineered to function across different size scales, from mm-sized tissues to 384-well plate format with automated liquid handling. The versatile applicability and potential of this platform for tissue engineering and organoid research were validated through successful differentiation into all three germ layers. Additionally, iPSC-microgel scaffolds of varying size scales were differentiated into functionally beating cardiac spheroids, further underlining the potential of this method. The reorganization potential and dynamic capabilities of the system were subsequently tested by introducing strategies towards prevascularization to increase the physiological relevance of the cardiac spheroid platform. The prevascularization strategies analysed included growth factor (GF)-guided co-differentiation and external introduction of endothelial cells from different origins. To demonstrate another possible application and support the versatility of the system, it was utilized for the investigation of subtype-specific developmental differences and requirements in biomaterial design between motor and sensory neuron lineages. Therefore, different biofunctionalizations and material introduction time points were tested, revealing the intrinsic differences between different neuronal subtypes during development and maturation, underlining the importance of regarding different neurons for spinal cord research. Concerning previous and ongoing work towards spinal cord regeneration in the group, as well as to compare the macroporous platform with a nanoporous PEG-hydrogel system, motor and sensory neuron spheroid differentiation inside a PEG-QK hydrogel was developed. The protocol and hydrogel parameters were successfully investigated, enabling the comparison of neuron development and neurite extension of motor and sensory neuron spheroids within the 3D matrix. Additionally, anisotropy was introduced into the system via the Anisogel platform, allowing for structural complexity and neurite guidance, important in the translational context of spinal cord regeneration.