3-D neural columns with tunable width and orientation

Organized three-dimensional (3-D) architectures exist in nature showing specific geometries, as cells can be randomly or anisotropically arranged. Since different tissues present different cell organizations, mechanical properties, and extracellular matrix (ECM) remodelling rates, controlled networks engineering is needed to tune specific in vitro tissues models, especially in 3-D1. While progress was made to align cells in two-dimensions (2-D), the reported fabrication techniques for aligned networks in 3-D are limited and don’t grant control over mechanical and porosity properties as revealed in nature2,3. Herein, we describe the use of a biomimetic ECM engineered system allowing fine control over mechanical and porosity properties of materials in 3-D, which can be programmed regarding stiffness, shape and directionality. Controlling polymeric ratios, and using crosslinking and freezing as a mechanism to guide porosity, hybrid 3-D structures are top-down fabricated with tunable micro-porosity to direct cellular responses. We demonstrated that crosslinking and temperature gradients can be used to direct network anisotropy, pore diameter, and structure stiffness with broad applications in modelling human tissues/interfaces, such as 3-D neural constructs. Using methacrylated gelatin and gellan gum as a hybrid polymeric blend and running a precise fabrication technique, we achieved in vitro anisotropic outgrowth of neurites as it is a unique characteristic in the brain cortex.