Tight junction formation of 2D enteroids. 2D enteroids grown on Matrigel coated A cell culture plastic or B, C transwells. Cells are stained after 3 days in culture for ZO1 (red) expression with a low power image (A) and inset magnified. Nuclei are stained with DAPI (blue). Representative images of four independent experiments. Scale bar: 30 µM. B Paracellular transport measured by diffusion of 4 kDa FITC-dextran across the 2D enteroids from the apical to basal compartment in Matrigel coated transwells. Measurements were taken at 0.5, 1, 2, 3, 4 h post-application of FITC-dextran to the apical chamber. To allow for normalisation across all time points, fluorescence measurements included a sample of cell culture media only. Data is converted to percentage maximum diffusion, measured in wells with Matrigel coated inserts and no cells. ***Represents statistically significant difference between Matrigel coated wells with cells and with no cells. Data is the median of four independent experiments and 95% CI. (C) Measurement of trans-epithelial electrical resistance (TEER) reaching maximum TEER at day 3 and maintained until day 7 of culture. Data is represented as median of four independent experiments and 95% CI.