Controlling the Organization and Differentiation of Human Neural Stem Cells on Hilbert Microcapillary Scaffolds Fabricated via Two-Photon Lithography.

3D scaffold architecture is critical for directing human neural stem cell (hNSC) fate and spatial organization. In this study, two-photon lithography (TPL) is used to fabricate microcapillary scaffolds based on the Hilbert space-filling curve as biomimetic basement membrane structures for guiding hippocampal-derived hNSC differentiation. The scaffolds feature 80 µm lumens with porous ellipsoidal membranes suspended above the substrate to provide topographical cues and permit nutrient diffusion while maintaining mechanical stability. hNSCs are cultured for 14 and 28 days and assessed via immunostaining for βIII-tubulin, nestin, GFAP, and synaptophysin. Confocal and electron microscopy reveal that scaffold geometry influenced both cell fate and spatial distribution: neurons aligned along the capillary membrane forming interconnected networks, while astrocytes extended projections across suspended support beams toward the membrane. Nestin expression remains elevated within scaffolds, suggesting a prolonged differentiation window relative to adjacent flat control surfaces. While this study does not include active perfusion or endothelial co-culture, the scaffold's accessible lumen geometry establishes a foundation for future neurovascular modeling. These results demonstrate that TPL-fabricated Hilbert scaffolds create structured microenvironments that modulate hNSC behavior, providing a reproducible and tunable platform for brain tissue engineering, drug screening, and mechanistic studies of neural development.