Abstract
Bioengineered 3D tunable neuronal constructs are a versatile platform for studying neuronal network functions, offering numerous advantages over existing technologies and providing for the discovery of new biological insights. Functional neural networks can be evaluated using calcium imaging and quantitatively described using network science. This protocol includes instructions for fabricating protein-based composite scaffolds, 3D in vitro culture of embryonic mouse cortical neurons, virally induced expression of GCaMP6f, wide-field calcium imaging, and computational analysis with open-source software and custom MATLAB code. For complete details on the use and execution of this protocol, please refer to Dingle et al. (2020).