Sequential DLP Bioprinting of Dual-Layered Brain Organoid-Like Neural Microtissues Using Nanocomposite Bioinks

This chapter provides a detailed, step-by-step protocol for the fabrication of architecturally defined brain organoid-like neural microtissues using a sequential digital light processing (DLP) bioprinting strategy. The method relies exclusively on DLP technology to fabricate complex heterogeneous structures through a layer-by-layer vat-switching technique. Central to this protocol is the formulation of a nanocomposite bioink, where reduced graphene oxide (rGO) nanoparticles and bacterial cellulose are mixed with gelatin methacryloyl (GelMA) matrix. This composition provides enhanced electrical conductivity, printability, and mechanical stability, promoting neural network maturation. We describe the complete workflow from induced pluripotent stem cell (iPSC) culture and neural induction, through bioink preparation and DLP printing parameter optimization, to long-term static culture and functional validation. This protocol addresses key limitations of conventional self-assembly methods, offering improved reproducibility, structural control, and physiological relevance for modeling neurodevelopment and disease.