As the human population ages the case of brain degenerative diseases will rise. The current treatment has a momentary effect and lacks an investigative system that is functionally applicable for testing. Though an optogenetic model and in vitro disease require a three-dimensional microenvironment, there is evidence that optogenetic stimulation is a viable approach. Differentiated SHSY-5Y cells were encapsulated in a 3D hydrogel (Gelatin Methacrylate) matrix in a multiplex system to examine the effects of optogenetic stimulation on neural development. SHSY-5Y cells are a human neuroblastoma cell line with characteristics similar to those of neurons when they are subjected to differentiation. Firstly, SHSY-5Y cells were genetically modified by tagging them with AAV “Optopatch” to express the light-gated ion channel channelrhodopsin-2 (ChR2)”, which causes depolarization in response to blue and red light stimulation. Complying with a 3D multiplex hydrogel scaffold, these modified cells were added, creating an environment conducive to the formation and development of neurons.ChR2-expression was stimulated by SHSY-5Y cells in the hydrogel constructions by applying precise blue light pulses using an optical setup. Assessing variables like calcium influx, neurite outgrowth, and synaptic activity, was tracked by the response of activated cells. Upon receiving optical stimulation, action potentials were generated. Moreover, synchronized activity between activated cells was shown by calcium imaging, indicating the development of functioning neural networks inside the hydrogel matrix. After successfully undergoing genetic modification and encapsulation, the neurons produced from human SH-SY5Y cells survived and expressed ChR2 in a Multiplex hydrogel system with Gelatin Methacrylate.