Morphological and electrophysiological properties of neural cells are substantially influenced by their immediate extracellular surroundings, yet the features of this environment are difficult to mimic in vitro. Therefore, there is a tremendous need to develop a new generation of culture systems that more closely model the complexity of nervous tissue. To this end, we engineered novel electrophysiologically active 3D neural constructs composed of neurons and astrocytes within a bioactive

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... llular matrix-based scaffold. Neurons within these constructs exhibited extensive 3D neurite outgrowth, expressed mature neuron-specific cytoskeletal proteins, and remained viable for several weeks. Moreover, neurons assumed complex 3D morphologies with rich neurite arborization and clear indications of network connectivity, including synaptic junctures. Furthermore, we modified whole-cell patch clamp techniques to permit electrophysiological probing of neurons deep within the 3D constructs, revealing that these neurons displayed both spontaneous and evoked electrophysiological action potentials and exhibited functional synapse formation and network properties. This is the first report of individual patch clamp recordings of neurons deep within 3D scaffolds. These tissue engineered cellular constructs provide an innovative platform for neurobiological and electrophysiological investigations, serving as an important step towards the development of more physiologically relevant neural tissue models.