Methods for a bioengineered 3D human brain-like tissue model of neuroregeneration after traumatic brain injury

Traumatic brain injury causes permanent cell death and can lead to long-term cognitive dysfunction, with no available treatments to repair the damaged brain tissue. Methods to track and understand traumatic brain injury in humans are severely limited by the inaccessibility of living brain tissue, creating a need for in vitro model systems to study cellular mechanisms of degeneration and regeneration following injury. Here we describe methods to establish a 3D human brain tissue model, consisting of a silk-collagen composite scaffold seeded with human neurons, astrocytes, and microglia, to study neuro-regeneration after traumatic brain injury. Step-by-step fabrication, injury, and analytical assessments of the 3D "triculture" system are described. Using this tissue model system, we demonstrate that glial cells promote regeneration of neuronal networks within the injury site over several weeks post-injury. Further, we found that regenerating networks in the 3D triculture tissues did not secrete early markers of neurodegenerative disease, but displayed signs of excitatory/inhibitory imbalance, suggesting that pro-regenerative treatments for traumatic brain injury in the future may need to direct cell differentiation to promote proper function. The mechanical stability of this model system enables physiologically relevant impact injury and long-term culture capability, while its modular design enables modification of cell contents, extracellular matrix composition, and scaffold properties. This adaptability could allow the integration of patient-derived cells and genetic modifications to bridge research and clinical applications focused on personalized targeted therapies. This in vitro system provides a valuable platform for accelerating therapeutic advancements in traumatic brain injury and neurodegenerative disorders, ultimately improving patient outcomes.