## 3 Results
### Barrier Device Design
#### 3.1.2 Channel Dimensions  
Channel lengths:  
For biological experiments, enough cells are needed to allow representative population-based analyses, and this implies a larger channel culture region. The channel lengths were adapted to take advantage of the entire length of a classic microscope slide (corresponding to the width of a classic plate). Therefore, the length of the upper channel was defined at 63 mm for a sufficient cell population. The contact surface between the two channels was made as large as possible so that most of the seeded cells should be in contact with the lower compartment. The two compartments can exchange along 39.4 mm, representing more than 62% of the cell monolayer. Channel width (\(w\)):  
The particularity of channel-based devices, in contrast to conventional wells and Transwell(r), is the higher edge area compared to low volumes. The geometry of the cell culture region highly influences the flow profile. In a non-cylindrical channel, the height-to-width (\(h/w\)) ratio of the cross section is an important consideration because it impacts on the shear stress distribution on the cell monolayer. When the medium flows in a rectangular channel, the velocity tends towards zero at the walls. This phenomenon generates two main issues. First, the cells growing near the side walls undergo a lower shear stress than the cells at the middle of the channel, which could consequently induce differences in cell behavior and measured permeability. Moreover, we may fear a longer retention time of paracrine signaling agents and analytes for permeability measurements at the walls. To limit these effects, the width of the channel cross section (\(w\)) must be much greater than its height (\(h\)) for a minimal \(h/w\) ratio [22; 23]. In our configuration, the thickness of PMMA sheets determines the channel heights. Lower available thicknesses are 0.3, 0.5, and 0.6 mm, so we defined the width \(w\) at 5 mm. Therefore, the \(h/w\) ratio was included between 0.06 and 0.12. This parameter is very important to achieve the most uniform shear stress across the entire cell surface. The uniformity of the shear stress distribution in our channel is analyzed below. With this length and width, we obtained a seeding area of 3.26 cm\({}^{2}\), allowing to have enough cells for permeability studies or genetic material extraction for expression analysis, for example. Upper channel height (\(h\)):  
Our workflow consisted of growing the cells in static condition for further days until confluence, before connecting the channel to a pump system for medium recirculation. In classic flasks and wells, large volumes of media limit the depletion of growth factors and nutrients contained in the medium, and dilute the waste products generated by cells. At the microscale, the channels have restricted dimensions, thus reducing the volume of medium above the cell surface. Consequently, the medium must be replenished more regularly to ensure nutrient supplying and waste elimination over the culture time in static. To measure the necessary volume of media for our cells, we applied the concept of \"effective culture volume and time\" introduced by [PERSON] et al. [24]. During the culture of hCMEC/D3 cells in flasks, we classically use 200 \(\mu\)L/cm\({}^{2}\) of medium (i.e., 5 mL in a T25 format or 15 mL in a T75 format), with a replenishment every four days. Consequently, cells use 50 \(\mu\)L per cm\({}^{2}\) per day. According to the previous defined length and width, the area of cell seeding represents 3.26 cm\({}^{2}\) in our device. In order to have 50 \(\mu\)L of medium per cm\({}^{2}\) and per day, the channel must contain 163 \(\mu\)L of medium changed every day. This results in a volume of 163 cm\({}^{3}\) on 3.26 cm\({}^{2}\) of the cell surface, so the channel height (\(h\)) must be 0.5 mm. If we chose a smaller \(h\), the volume medium would be insufficient for one day of supply, so we would have to change the medium more frequently than once a day.