## 4 Discussion
The choice of a parallel static control is important for comparison to the fluidic condition, but is often lacking due to the device characteristics [31; 32; 35; 39; 52; 53]. In some cases, Transwell(r) assays are used but do not represent an accurate control because of large differences in terms of format, material, and surface area to volume ratio [34; 37; 54; 57]. In addition, the duration of the culture and the medium exchange have to be identical for a suitable comparison. To overcome all these points, we created a device for culturing BMEC under physiological shear stress to help their differentiation, and characterize them before performing permeability studies. Two vertically superimposed channels were designed for direct visual observation at any time of the culture. The channel lengths were defined to implement a large exchange surface between both compartments. It provides sufficient volumes for sampling during experiments, but also to collect cells or genetic material for further analysis. The heights of the channels were adapted for easy cell maintenance, and to allow a direct characterization of BBB markers by confocal microscopy. The width of the channels ensures a physiological shear stress homogenously distributed along the channel. Our device configuration allows an easy cell culture of any cell type, both in the upper and/or lower channels. Here, we seeded human cerebral endothelial (hCMEC/D3) cells on the porous membrane at the interface between the channels. The barrier device is compatible with any pump system and can support a wide range of shear stresses (including physiological values) while ensuring laminarity. All along the culture, optical microscopy was used to monitor cellular organization and confluence. We confirm the hCMEC/D3 cell realignment in the flow direction, and demonstrate that culture in the static condition is also suitable with a daily renewal of culture medium. BMEC characterization was studied directly in the device by immunodetection of both tight and adherent junctions at cell-to-cell boundaries. As for quality control, cells can also be extracted to isolate nucleic acids or proteins for further exploration of specific BBB markers in both conditions. Finally, our barrier device is expected to elucidate mechanisms of drug transport at the BBB. The large membrane interface combined with the low channel volumes allow an easy detection of compounds. Here, the barrier selectivity for the LY paracellular marker was reported as an example to confirm the feasibility of permeability studies in these channels. The plate format offers the possibility to use six replicates simultaneously in the same environment, or to create different individual experimental conditions in parallel. Finally, the cost of our barrier device is affordable as it represents less than six euros per condition. Limitations and future works:  
Some points still deserve further investigations to achieve a complete characterization of this model. Firstly, the device configuration offers the possibility to incorporate other cell types in the lower compartment for a more physiologically relevant model. Co-culture induces cell-cell interactions for further BMEC differentiation towards a BBB phenotype [58]. Among the cellular components of the BBB, glial cells are known to particularly improve the function and regulation of BMEC [59; 60]. However, in hCMEC/D3 cells specifically, improvement of BBB properties in response to astrocytes was controversial since two studies reported no changes in endothelial cell function [43; 61]. For this reason, and to firstly develop a simple model, we preferred to validate our device with a monoculture. However, in future works, it could be interesting to test this configuration to rule on the co-culture impact on hCMEC/D3 cells in combination with shear stress. Analysis of cell orientation was performed to validate the effective shear stress exposure. Nitric oxide is another indicator that could also be used.