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.
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Highlights • In longer-term culture, granular hydrogels supported the development of neural culture with extensive neurite outgrowth. • Granular hydrogels supported significantly higher cell viability and neurite outgrowth over 7 days of culture. • A simple 3D printed extrusion and mixing toolset is used to generate and homogenously seed cells into granular hydrogels. • The toolset designs are provided for 3D printing, enabling use and customization of the techniques described.