2.3. Cell seeding and co-culture on the chip and wells
The upper gut barrier module has 0.2×0.2 mm channel size, 0.5 cm2 cell culture area and capacity of 0.6 ml medium. The bottom BBB module has 0.3×0.3 mm channel size, 0.28 cm2 cell culture area and capacity of 0.85 ml medium. A 96 well plate (Corning, USA, 3988) and 24 transwell plate (Corning, USA, 3470) were used for control experiment. Each well of 96 well plate contains 0.3 ml of medium, each apical chamber of 24 well plate contains 0.2 ml of medium, and each basal chamber of 24 well plate contains 1 ml of medium.
The brain endothelial cell line was used first to optimize initial seeding condition for later experiments using hBMECs. Caco-2 was suspended in 0.1 ml of medium at 2×106 cells/ml density to be seeded at 4×105cells/cm2 on upper module, cultured for 5 days until cells completely cover the membrane, and exposed to fluidic flow for next 5 days. 2 days after Caco-2 was exposed to the flow, either bEnd.3 or hBMECs was were suspended in 0.056 ml of medium at 3×105 cells/ml density to be seeded at 6×104 cells/cm2 on each bottom module. After culturing the endothelial cells for 3 days, the modules were assembled and co-cultured under flow condition for 24h. For control experiment, gut cells and brain endothelial cells were cultured in wells at same cell density.
Lipopolysaccharide (LPS; Sigma, USA, L2637) and sodium butyrate (NaB; Sigma, USA, B5887) were chosen as a model toxin and a model drug. 100 μg/ml of LPS was treated onto the gut in both well and chip while brain endothelial cells in well were treated with 0.1 μg/ml of LPS, for 24 hours in all cases. 2 mM of NaB was treated onto gut cells in the chip and 0.2 mM of NaB was treated onto brain endothelial cells in transwell for 24 hours.