Increased connectivity of hiPSC-derived neural networks in multiphase granular hydrogel scaffolds

To reflect human development, it is critical to create a substrate that can support long-term cell survival, differentiation, and maturation. Hydrogels are promising materials for 3D cultures. However, a bulk structure consisting of dense polymer networks often leads to suboptimal microenvironments that impedes nutrient exchange and cell-to-cell interaction. Herein, granular hydrogel-based scaffolds were used to support 3D human induced pluripotent stem cell (hiPSC)-derived neural networks. A custom designed 3D printed toolset was developed to extrude hyaluronic acid hydrogel through a porous nylon fabric to generate hydrogel granules. Cells and hydrogel granules were combined using a weaker secondary gelation step, forming self-supporting cell laden scaffolds. At three and seven days, granular scaffolds supported higher cell viability compared to bulk hydrogels, whereas granular scaffolds supported more neurite bearing cells and longer neurite extensions (65.52 ± 11.59 μm) after seven days compared to bulk hydrogels (22.90 ± 4.70 μm). Long-term (three-month) cultures of clinically relevant hiPSC-derived neural cells in granular hydrogels supported well established neuronal and astrocytic colonies and a high level of neurite extension both inside and beyond the scaffold. This approach is significant as it provides a simple, rapid and efficient way to achieve a tissue-relevant granular structure within hydrogel cultures

Engineering human neural networks: controlling cell patterning and connectivity

Restorative treatments for diseases affecting the central nervous system (CNS) are difficult to develop, due to the complexity of CNS tissues and transferability issues with results from costly animal models. There is an urgent need to produce reliable, complex culture models to develop effective treatments. Such models require controlled interfaces and relevant dense neural cultures. In this thesis, techniques are developed to prepare and investigate scaffolds enabling complex structured neural model fabrication. To facilitate development of an interface layer, neural culture on polycarbonate track-etched membranes demonstrated that the growth of neural processes through pores required confluent cultures. To direct low density cultures, funnel-shaped pores were machined into glass coverslips, and neurite interaction with angled pore edges was analysed. Live-imaging results showed that neurites more often crossed shallower edges, and retreated from steeper edges. Concerning development of dense cultures, neural culture in non-granular hyaluronic acid (HA) hydrogels showed cell clustering and reduced neurite extension. A protocol adding secondary structure to the scaffold by granulating HA hydrogel was optimized, and cell viability and connectivity within the hydrogel were analysed. Cell viability in the granular hydrogel was comparable to the control, and there was improvement of network connectivity in granular hydrogels over non-granular counterparts. Potential application to improve nerve graft technology motivated the design of an extrusion device that generates tertiary structure by interspersing cell-seeded and unseeded granular HA hydrogel, facilitating control of cell distribution and alignment within the scaffold. Tertiary extruded and non-extruded hydrogels were analysed, and distribution was maintained within the tertiary extruded hydrogel scaffold, without detriment to the cell functionality. It is hypothesized that additional guidance cues could be added to the scaffolds to control cellular alignment. Findings demonstrate the fabrication of structured scaffolds optimized for neural network growth, and highlight strategies that can be used in the production of in vitro neural models for complex CNS study.</p

Engineering human neural networks: controlling cell patterning and connectivity

Restorative treatments for diseases affecting the central nervous system (CNS) are difficult to develop, due to the complexity of CNS tissues and transferability issues with results from costly animal models. There is an urgent need to produce reliable, complex culture models to develop effective treatments. Such models require controlled interfaces and relevant dense neural cultures. In this thesis, techniques are developed to prepare and investigate scaffolds enabling complex structured neural model fabrication. To facilitate development of an interface layer, neural culture on polycarbonate track-etched membranes demonstrated that the growth of neural processes through pores required confluent cultures. To direct low density cultures, funnel-shaped pores were machined into glass coverslips, and neurite interaction with angled pore edges was analysed. Live-imaging results showed that neurites more often crossed shallower edges, and retreated from steeper edges. Concerning development of dense cultures, neural culture in non-granular hyaluronic acid (HA) hydrogels showed cell clustering and reduced neurite extension. A protocol adding secondary structure to the scaffold by granulating HA hydrogel was optimized, and cell viability and connectivity within the hydrogel were analysed. Cell viability in the granular hydrogel was comparable to the control, and there was improvement of network connectivity in granular hydrogels over non-granular counterparts. Potential application to improve nerve graft technology motivated the design of an extrusion device that generates tertiary structure by interspersing cell-seeded and unseeded granular HA hydrogel, facilitating control of cell distribution and alignment within the scaffold. Tertiary extruded and non-extruded hydrogels were analysed, and distribution was maintained within the tertiary extruded hydrogel scaffold, without detriment to the cell functionality. It is hypothesized that additional guidance cues could be added to the scaffolds to control cellular alignment. Findings demonstrate the fabrication of structured scaffolds optimized for neural network growth, and highlight strategies that can be used in the production of in vitro neural models for complex CNS study.</p