Development of a 3D Neurovascular Unit for In Vitro Modeling of Neuropathologies and Drug Discovery

Introduction:: Introduction: The human brain has complex physiology that poses a substantial challenge for developing anatomically and physiologically relevant models of human brain pathologies in animals and in vitro. Consequently, animal models used to screen potential therapeutics in vivo are not able to provide essential information regarding the interactions between the therapeutic and the human-specific physiology. This knowledge gap contributes to widespread clinical trial failures. These failures and the lack of in vitro platforms that accurately represent the human in vivo niche highlight an urgent need for technologies mimicking complex brain anatomy, physiology, and functions to study mechanistic interactions in drug discovery. To address these limitations, we have developed a perfusable, fully vascularized and anatomically correct three-dimensional (3D) model of the NVU in a perfusable microfluidic chip using ex vivo human tissue embedded in a biomimetic hydrogel containing the N-Cadherin adhesion motif. We verified the structural hierarchy of all cells required to create a perfusable vascular network. Overall, our 3D NVU model provides a more anatomically accurate system that allows for dynamic interrogation of the NVU and BBB in healthy and diseased tissue.


Materials and Methods:: Materials and Methods: Microfluidic devices were fabricated using a stereolithographic 3D printed mold. This mold was vapor deposited with Parylene C to provide a physical barrier to prevent cytotoxic leeching into the casted polydimethylsiloxane and hydrogel. Fresh human brain tissue was collected and minced in a 1mL conical tube filled with 500µL of Hank’s Balanced Salt solution for 5 minutes. The vial was then centrifuged at 2500 rpm. After centrifugation, the vial was tilted and the liquid top layer was aspirated. Then 100µL of minced ex vivo tissue was transferred to 1mL of 10% hydrogel and thoroughly mixed. Subsequently, microbial transglutaminase was added to the hydrogel for crosslinking (2% final solution), mixed and poured into the microfluidic device. The hydrogel was cured for 30 minutes at 37°C, followed by immediate perfusion of the peripheral channels. Vascular growth in the hydrogels was imaged with confocal immunofluorescence 7 days following tissue embedding.


Results, Conclusions, and Discussions:: Results and Discussion: We established a biomimetic hydrogel that supports iPSC-derived glutamatergic neurons, glial and mural cells in 3D (Figure 1). Additionally, our biomimetic hydrogel promotes arteriogenesis of ex vivo human brain tissue (Figure 2), and we established a neurovascular in vitro model with the human brain tissue (Figure 3). The newly formed arterioles and capillaries formed a patent lumen with correct concentric spatial organization (claudin 5⁺ endothelial cells, collagen IV⁺ basement membrane, and smooth muscle actin⁺ cells/NG2⁺ pericytes) (Figure 2). Finally, highlighting a key feature of this novel ex vivo model, the newly grown capillary network demonstrated a robust and impermeable BBB integrity (Fig. 4). Diffusion and permeability coefficients were quantified, revealing barrier properties two orders of magnitude greater than models using immortalized cells^1,2.



Conclusions: As a proof of concept, we developed a neurovascular unit containing endothelial cells, pericytes and smooth muscle cells. We have shown that using ex vivo human brain tissue in our biomimetic hydrogel induces continuous vascular growth with anatomically correct hierarchy and expression of characteristic markers of vascular cell phenotypes. Finally, we demonstrated lumen-perfusion of this newly formed vascular network using fluorescent microbeads and demonstrated this ex vivo model develops a robust blood-brain barrier. We believe this platform represents a realistic biomimetic microenvironment that will allow for unprecedented access to not only gain a deeper understanding of disease progression and pathology, but gain the ability to spatially and temporally observe the interactions of multiple cell types involved in neuropathologies.


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References (Optional): : Osaki, T.; Sivathanu, V.; Kamm, R. D. Vascularized Microfluidic Organ-Chips for Drug Screening, Disease Models and Tissue Engineering. Curr. Opin. Biotechnol. 2018, 52, 116–123. https://doi.org/10.1016/j.copbio.2018.03.011.

Bhalerao, A.; Sivandzade, F.; Archie, S. R.; Chowdhury, E. A.; Noorani, B.; Cucullo, L. In Vitro Modeling of the Neurovascular Unit: Advances in the Field. Fluids Barriers CNS 2020, 17 (1), 22. https://doi.org/10.1186/s12987-020-00183-7.