3D in vitro modeling of neural microenvironment through a multi-scaffold assembly approach

The engineering of in vitro 3D cell culture systems has emerged as promising approach to model central nervous system (CNS) intricacy with increasing physiological relevance. The fabrication of artificial microenvironments that closely resemble nervous tissue composition and architecture has provided useful substrates to promote neural cell growth and maturation under in vivo-like conditions; however, despite significant progress has been made in tissue mimicry, directing neural cell arrangement and connectivity in a controlled 3D environment remains extremely challenging. Here, we propose a novel approach that combines different biomaterials and biofabrication techniques to develop a multi-scaffold system mimicking distinctive features of the nervous tissue. Extrusion-based 3D bioprinting is employed to accurately position neural stem cells (NSCs) embedded in a gelatin methacryloyl hydrogel onto an aligned microfibrous polycaprolactone structure obtained by melt electrowriting. The hydrogel matrix successfully supports NSC growth within 3D bioprinted constructs, ensuring high cell viability and in situ NSC differentiation into neuronal and glial phenotypes. Additionally, melt electrowriting technology allows the design of a microfibrous scaffold having well-defined geometry and aligned microporosity to replicate the anisotropic characteristics of nervous tissue. The inclusion of such scaffold in the 3D bioprinted system effectively steers neural cell organization in a 3D setting, guiding neural cell elongation in a preferred direction and promoting the establishment of a functional neural network. Our approach can be used to develop more sophisticated multicellular systems, possibly reassembling specific CNS circuits within a biomimetic microarchitecture, thus offering a versatile platform for the investigation of CNS functioning and pathology.