A Long-Lived Human Neurovascular PENTA Culture Model Captures Incomplete Vascular Repair and Glia-Associated Signaling After Traumatic Brain Injury.

Traumatic brain injury (TBI) frequently leads to chronic neurovascular dysfunction, yet mechanistic insights into human-specific responses have been limited by the absence of long-term, multicellular in vitro models. Here, we report a five-cell-type human neurovascular culture system, comprising endothelial cells, astrocytes, pericytes, microglia, and neurons, engineered within a 3D scaffold to study injury-induced remodeling over multiple weeks. This PENTA-culture platform captures key structural and molecular features of the neurovascular unit and supports compartment-resolved profiling of vascular and neuroimmune responses. Under baseline conditions, PENTA cultures exhibit restricted tracer distribution relative to simpler culture configurations, consistent with the emergence of barrier-like properties within the 3D scaffold. Following mechanical trauma, cultures exhibit a biphasic response characterized by acute endothelial disorganization, mitochondrial structural changes, and neuroimmune alterations, followed by delayed and incomplete structural recovery, accompanied by shifts in angiogenic and immunomodulatory signaling consistent with Tyrosine kinase with immunoglobulin-like epidermal growth factor-like domains 2 (Tie2)- and Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT)-associated signatures. At last, the inclusion of microglia and neurons is associated with improved cytokine resolution and partial recovery of junctional organization, highlighting the influence of neuroimmune complexity on post-injury vascular remodeling. Together, this long-lived, human-derived platform provides a structurally complex and functionally informative system for characterizing neurovascular injury signatures following TBI.