## 4 Discussion  
In this study, we present the creation of a high-throughput microfluidic device based on an in vitro model of the human blood-brain barrier (BBB) [21,29]. The patterning of ECM is made possible by the surface tension of the microfluidic platform. A BBB microvessel forms in the perfusion channel next to the ECM. The technique enables fluid-phase sampling of molecules that flow through the endothelium and extracellular matrix layers, allowing fluid to flow through BBB microvessels without the need for artificial membranes. We demonstrate that the model is sensitive to variations in A\(\beta\) concentrations. The focus of the paper is to investigate the transportation of A\(\beta\) oligomers across the blood-brain barrier (BBB), specifically examining A\(\beta\) accumulation and penetration at the BBB. This study has the potential to pave the way for future research to explore the role of A\(\beta\) transporters such as RAGE and LRP1 in mediating A\(\beta\) transport across the BBB. It is worth noting that the cells utilized in this model are immortalized, they may not have the same phenotype as BBB cells found in native cells. As a result, patient outcomes from this model might not be applicable directly. Nonetheless, regardless of the passage number, it has been demonstrated that the endothelial cell line in this investigation expresses tight junctional markers and forms BBB [30, 31, 32, 33, 34]. This paper examined the effect of A\(\beta_{1-42}\) oligomer on the BBB grown on a membrane-free microfluidics device. We investigated (1) the integrity of the BBB, (2) the expression of ZO-1 tight junctional protein, and (3) cell viability before and after exposure to A\(\beta_{1-42}\) solutions. In the microfluidic device, the endothelial layer formed the 3D tubular structure against the membrane-free ECM, which provides a physiologically relevant exposure route for A\(\beta_{1-42}\). To determine the barrier integrity of the BBB, FITC was infused into the BBB cultures within the microfluidic device. Time-lapse photography was used to track FITC penetration in the ECM lane, and the apparent permeability (\(\mathbf{P}_{app}\)) was estimated. The same method was applied to determine \(\mathbf{P}_{app}\) in chips that had or did not have an A\(\beta_{1-42}\)-treated endothelial microvessel. Our findings suggest that FITC diffuses through the ECM to the same degree whether the barrier is absent or the BBB has been modified with A\(\beta\). The results demonstrate the increase in FITC permeability across the BBB with the increase in A\(\beta_{1-42}\) concentrations (Figure 3). Figure 5: Cell viability assay of endothelial cells seeded on the two-lane microfluidics. (**A**) A representative series of confocal microscopy images depicting live/dead staining of endothelial tubules in microfluidic channels. Green fluorescence indicates live cells and red fluorescence indicates dead cells. (**B**) Percentage of live/dead 48 h following treatment with amyloid beta (A\(\beta_{1-42}\)). All data represent average viability measured within the top and bottom layers of the endothelial tubule. The value represents the mean \(\pm\) SD for two independent experiments (n = 2 for each treatment condition). Interestingly, we observed that an increase in A\(\beta_{1-42}\) concentration was associated with an increase in the intensity of ZO-1 (Figure 4D,E). We believe that, when endothelial cells were treated with 20-30 uM A\(\beta_{1-42}\), A\(\beta\) was accumulated on the wall of ECs against the ECM and formed cell-A\(\beta\) matrix aggregates, eventually forming the aggregate of dysfunctional BBB (Figure 6). It seems as though the complex cell-A\(\beta\) matrix does not allow A\(\beta\) penetration into the brain parenchyma (ECM channel/brain lane), with the A\(\beta\) intensity in the ECM decreasing with increasing A\(\beta\) concentrations (Figure 4C).