Fabrication of 3D monolayer nanofiber structures via capillary induced bending and fusion

Electrospun nanofibers offer tunable topography and broad material versatility for advanced cell culture studies and cell-based applications; however, the fabrication of free-standing, highly porous, and ultrathin nanofiber architectures remains challenging. Here, we report a solvent evaporation and capillary-force-assisted fusion of two monolayers of gelatin nanofibers deposited on opposite sides of a through-hole membrane. During solvent evaporation, capillary forces drive the two nanofiber layers into contact in each hole, enabling localized nanofiber fusion in parallel of chemical crosslinking. This process yields an array of microwells with three-dimensional (3D) monolayer nanofiber features in each well. We quantitatively analyzed the fusion dynamics and elucidate the interplay among capillary pressure, membrane bending, and boundary conditions. The resulting nanofiber features exhibit robust mechanical integrity, arising from the entanglement of crosslinked nanofibers and their anchoring within the membrane through-holes. As a demonstration, these structures function as biologically relevant platforms for 3D cell culture and co-culture studies. Overall, this capillary-force-assisted nanofiber processing is simple yet versatile, offering a scalable route toward large-area nanofiber-based biointerfaces.