Engineering 3D microenvironments with femtosecond laser-fabricated microtubes to direct neuronal network formation

Abstract

The regulation of cell growth characteristics through physical structures on material surfaces plays a crucial role in advancing our understanding of cell behavior. Three-dimensional (3D) microstructures, offering multidimensional growth space and a richer nutrient environment compared to their two-dimensional counterparts, have attracted significant attention from researchers. However, the fabrication of high-precision 3D microstructures, especially those with a high aspect ratio (H-AR), remains challenging. In this study, we employ femtosecond laser to achieve exceptional precision and patterning capabilities in fabricating 3D microstructures. To enhance the manufacturing efficiency of axial 3D structures, we introduce a novel single pulse multi-photon polymerization (SP-MPP) method for efficient fabrication of H-AR micropillar and microtube structures. Our innovative approach involves a dual-aperture optical modulation method that effectively modulates the annular energy distribution, enabling the creation of H-AR microstructures. We can achieve a maximum processing height of up to 130 μm with an aspect ratio of 16 within single pulse exposure of 217 fs. Compared to the layer-by-layer scanning method, our proposed approach enhances the axial manufacturing efficiency of 3D structures by 1–2 orders of magnitude. Through experimentation, we validate the applicability of microtube structures for guiding neuronal orientation by utilizing microtube arrays in hippocampal neuronal cell culture. Our results demonstrate that neurons not only grow along the patterned microtubes but also exhibit preliminary synaptic differentiation. Furthermore, we effectively regulate the 3D patterning of neuronal growth by implementing diverse patterned microtube arrays. This proposed method enables the rapid construction of a large-scale 3D cell growth environment, offering significant advantages in inducing neuronal orientation. Consequently, this technique holds immense potential for diverse applications in biomedicine and tissue engineering, including the establishment of isolated neural networks.