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Neural Engineering

Neural Engineering - Poster Session B

Poster K8 - Development of 3D In-Vitro Model of the Human Neurovascular Unit to Study Traumatic Brain Injury

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Traumatic brain injury (TBI) is characterized by an initial mechanical injury, which launches a complex cascade of poorly understood molecular and metabolic events which are amplified by interactions between the different cells in the brain. Recent studies demonstrated a link between mild repetitive TBI (mrTBI) and the progression of a neurodegenerative tauopathy called chronic traumatic encephalopathy (CTE). The hallmark of CTE pathology is the accumulation of hyper-phosphorylated tau proteins around neuronal and vascular networks at the depths of sulci. The human neurovascular component (NVU) plays a critical role in the onset and progression of this disease; however, the exact mechanisms remain unknown due to the lack of a physiologically relevant model that can replicate the CTE phenotype. Thus, here, we developed a human 3D in vitro model of the neurovascular unit to study the molecular mechanisms of mrTBI-induced neurodegeneration

Materials and

Methods:  Our NVU model is comprised of 3 main cell types: green fluorescent protein-expressing human brain microvascular endothelial cells (GFP-hBMEC) (E), astrocytes (A), and pericytes (P) seeded into porous silk scaffolds at a ratio of 1:0.5:0.3 million cells and enveloped into collagen type I gel. Tested conditions (1) Extracellular matrix (ECM): laminin, fibronectin, collagen type 4, and Matrigel; (2) Cellular compositions: EAP, EP, and EA. All procedures were completed following the manufacturer’s specifications.

Results, Conclusions, and Discussions: We optimized the 3D in-vitro neurovascular unit (NVU) model along 3 main factors: cell density, extracellular matrix (ECM) supplementation, and cell composition. First, to select suitable cell densities of BMECs, we used 2, 1, and 0.5 million cells. 1 and 2mln of green fluorescent protein-expressing human BMECs (GFP-hBMEC) showed vessel-like and vascular network-like structure formation, observed via confocal and epifluorescent microscopy at 14- and 28-days post-seeding. Second, we used the following ECM components: laminin, fibronectin, collagen type 4, and Matrigel. We discovered that each ECM component positively affected the general cellular activity at 14 and 28 days; however, Matrigel had the strongest effect on vascular network formation. Third, various cellular compositions (EAP, EP, and EA) were used to assess how the multicellular interactions affect the formation of vessel-like and vascular network-like structures. Both co(di)-culture and tri-culture models showed improved vascular network-like structure and tight junction formation compared to cell density and ECM component experiment samples. Homeostatic growth of all cells was validated and confirmed by analyzing ZO1 and claudin-5 (tight junction markers), CD31 (BMEC functional marker), Aquaporin-4 (astrocyte marker), and α-Smooth Muscle Actin (pericyte) markers through western blot. Finally, mrTBI demonstrated a significant increase in hpTAU expression, altered mitochondria network integrity, driven by pDRP1-dependent fission, increased IL-1b signaling, and decreased tight junction markers short and long-term after injury.
Different seeding densities of hBMECs were studied, and 1 million cells/scaffold was chosen as it showed optimal endothelial cell growth yet had the potential for improvement. Extracellular matrix proteins drastically affected the network formation at early time points (up to 4 weeks), while after this point, the differences between ECM conditions and controls began to diminish. Co(di)-culture and tri-culture models with the combination of different cell types, including hBMEC, astrocytes, and pericytes, significantly boosted network formation at early time points, and these samples remained distinguished beyond two months. Moving forward, this model will be used with neuronal cells to study the molecular mechanisms that drive CTE-like phenotype in response to mild repetitive injuries.

Acknowledgements (Optional):  We thank the University of Cincinnati for its financial support to Dr. Liaudanskaya and the Cincinnati Children’s Hospital Bioimaging and Analysis Facility.