Fig. 1

Validation of glass cylinder methodology. A (i) 25 µL drop of trypan blue in PBS applied on top of the HEE, (ii) the basolateral penetration of trypan blue after 4 h of incubation (red arrow), and (iii) H&E staining showing the open edges of the HEE (black arrow). B (i) Glass cloning cylinder on top of the HEE indicated with the black arrow, (ii) 25 µL of trypan blue in PBS was pipetted inside the cylinder, (iii) the PBS was evaporated 4 h later (in flow cabinet on heated plate at 37 °C, without lid), and (iv) the removal of the cylinder revealed a blue colonized circle without basolateral penetration. C Lucifer yellow (LY) added inside the glass cylinder and harvested after 2.5 h of incubation. DAPI staining and fluorescent imaging (× 10 magnification) shows (i) the distribution of LY onto the whole HEE and (ii) clean edges. D H&E, Ki-67, and filaggrin (FLG) staining of HEE with a drop of PBS on top for 24 h to analyze the morphological changes and protein expression patterns compared to the control. E Difference in morphology between the removal of the cylinder after PBS evaporation or leaving it on top of the HEE for 48 h shown with an H&E staining. F Schematic overview of HEE culture and the topical application of bacteria using a glass cylinder. Scale bar = 100 µm