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.