Engineering geometrical 3-dimensional untethered in vitro neural tissue mimic

Significance Forward design of tissue-like structures is very important for biomedical and engineering applications. We developed a methodology for designing 3-dimensional (3D) neural tissue mimics that can be formed into desired shapes and sizes, while maintaining their electrical functionality, and can be physically transferred to different platforms. These biofabricated neural constructs could facilitate assembling biological machines, advance methods for assessment of neural functionality in vitro, and support the development of improved models for disease studies. Formation of tissue models in 3 dimensions is more effective in recapitulating structure and function compared to their 2-dimensional (2D) counterparts. Formation of 3D engineered tissue to control shape and size can have important implications in biomedical research and in engineering applications such as biological soft robotics. While neural spheroids routinely are created during differentiation processes, further geometric control of in vitro neural models has not been demonstrated. Here, we present an approach to form functional in vitro neural tissue mimic (NTM) of different shapes using stem cells, a fibrin matrix, and 3D printed molds. We used murine-derived embryonic stem cells for optimizing cell-seeding protocols, characterization of the resulting internal structure of the construct, and remodeling of the extracellular matrix, as well as validation of electrophysiological activity. Then, we used these findings to biofabricate these constructs using neurons derived from human embryonic stem cells. This method can provide a large degree of design flexibility for development of in vitro functional neural tissue models of varying forms for therapeutic biomedical research, drug discovery, and disease modeling, and engineering applications.