Enhancement of the Advanced Microphysiological Brain Injury Technology (AMBIT) into a Completely Xeno-Free Isogenic Multicellular Platform

Traumatic brain injury (TBI) is a significant health issue which affects service members and veterans during times of both peace and war. The high rate of TBI and blast-related concussion events resulting from current combat operations directly impacts the health and safety of individual service members and subsequently the level of unit readiness and troop retention.. The impacts of TBI are felt within each branch of service, including the Air Force (?13% from 2000-2020 Q3), and throughout the Department of Defense and the Department of Veterans Affairs health care system. TBI is closely associated with alterations of the blood-brain barrier (BBB). The BBB is a highly selective interface between the brain vasculature and neural tissue, comprised mainly of brain microvascular endothelial cells (BMECs), pericytes, astrocytes, microglia, and neurons and its integrity is crucial for proper brain structure and function. Although its disruption is a key consequence of TBI, the mechanisms behind this process are still poorly understood – mostly due to the lack of physiologically relevant BBB in vitro models for the purpose of studying long-term disease mechanisms, and as a platform for drug development, safety and efficacy testing. To address this gap we have developed AMBIT, a novel BBB-on-chip model that enables the compartmentalized co-culture of cell types present in the neurovasculature, incorporation of shear stress through fluid flow, and TBI modeling through high-speed stretch. The biocompatibility of the platform was initially validated using co- cultures of rat primary BMECs and astrocytes, followed by the incorporation of human immortalized BMECs (hCMECs/D3) and demonstration of their responsiveness to single and repetitive moderate TBIs. Currently, AMBIT includes the co-culture of human iPSC-derived BMECs (iBMECs) and brain pericytes (iPericytes) grown in opposite sides of the porous membrane, facing the vascular and neural compartments, respectively. The use of iPSC-derived cells not only allows AMBIT’s application in personalized medicine and drug screening, but also greatly increases the physiological relevance of our model. In addition to the use of human cells, our efforts to improve AMBIT’s ability to properly mimic the human neurovasculature have been focused on the removal of all animal-derived products from the system and adoption of fully defined components.