Stable
enteroid lines were
grown in matrigel for 5–7 days before breaking the spheres
using a G27 needle and a syringe. The single cell suspension was then
seeded onto well inserts (Corning Transwell clear polyester membrane
0.4 μm, 0.33 cm2, Sigma-Aldrich) coated with collagen
from human placenta (Sigma-Aldrich) and cultured in Intesticult (components
A and B, Stem Cell) for 5–10 days to form confluent monolayers.
The confluency was assessed using a microscope and measuring trans
epithelial resistance (TER) with a Millicell ERS-2 V/ohm meter (Millipore).
When TER reached above 600 Ω/cm2 (minus the well
background resistance), the cells were grown in equal parts Intesticult
component A + DMEM for 5 days to induce the differentiation of the
cells into a more mature state as previously described.42 (link) The differentiated monolayer cultures were then
subjected to an apical CT challenge (0.1 μg/mL) where CT had
been pretreated with polymers or oligosaccharides. To monitor the
CT-induced ion efflux from the TER cells, the trans epithelial resistance
and voltage were monitored, where the short circuit current (Isc) per cm2 was calculated. The results
were then normalized to the measurements on PBS-treated cells.
enteroid lines were
grown in matrigel for 5–7 days before breaking the spheres
using a G27 needle and a syringe. The single cell suspension was then
seeded onto well inserts (Corning Transwell clear polyester membrane
0.4 μm, 0.33 cm2, Sigma-Aldrich) coated with collagen
from human placenta (Sigma-Aldrich) and cultured in Intesticult (components
A and B, Stem Cell) for 5–10 days to form confluent monolayers.
The confluency was assessed using a microscope and measuring trans
epithelial resistance (TER) with a Millicell ERS-2 V/ohm meter (Millipore).
When TER reached above 600 Ω/cm2 (minus the well
background resistance), the cells were grown in equal parts Intesticult
component A + DMEM for 5 days to induce the differentiation of the
cells into a more mature state as previously described.42 (link) The differentiated monolayer cultures were then
subjected to an apical CT challenge (0.1 μg/mL) where CT had
been pretreated with polymers or oligosaccharides. To monitor the
CT-induced ion efflux from the TER cells, the trans epithelial resistance
and voltage were monitored, where the short circuit current (Isc) per cm2 was calculated. The results
were then normalized to the measurements on PBS-treated cells.
