Two-photon polymerization of hydrogel cellular scaffolds

Three-dimensional (3D) micro-nano fabrication through two-photon polymerization (TPP) offers a convenient way to fabricate complex structures with fine precision using hydrogel materials commonly employed in biological cell culture. Presently, tissue engineering scaffold fabrication encounters challenges related to processing precision and the creation of complex structures. We developed a 780 nm fs laser system integrated with a 2D galvanometer system and Z-axis to create a 3D micro-nano printing system. Arbitrarily complex structures that simulate the vascular environment in vivo can be fabricated, taking advantage of the fact that the femtosecond laser is focused only inside the photoresist. Through testing the linewidth resolution of GelMA hydrogel via two-photon polymerization processing, we identified optimal laser power, scanning rate, and photoinitiator concentration for precise processing. We successfully produced a simple tissue scaffold, a regular structure composed of several cubes. A complex structure scaffold simulating the structure of mouse blood vessels was printed, and we successfully seeded the mouse neuron cells on it. This confirms the feasibility of replicating the in vivo vascular environment in vitro and utilizing two-photon micro-nano-processed scaffold structures for cell culture experiments. The ability to fabricate complex microstructures with high precision and resolution using GelMA hydrogel opens up new possibilities in tissue engineering, regenerative medicine, and drug delivery systems. • Hydrogel GelMA 3D printed complex tissue scaffolds using TPP can mimic the in vivo environment for cell growth in vitro . • GelMA linewidth of 200 nm is fabricated using only a 20× objective lens with a low numerical aperture (NA = 0.4). • Provide a cost-efficient method for the fabrication of cell culture scaffolds.