Abstract

Barrier integrity is a core epithelial function of keen interest
for drug discovery. We established a scalable 3D quantitative immunofluorescent
imaging assay to evaluate junction complexes and combined it with TEER to
assess multiple barrier integrity mechanisms at once. Air-lifted human bronchial
epithelial cells had TEER measured pre- and post-treatment. Fixed cells were
stained for adherens (E-cadherin) and tight (ZO-1, occludin) junctions.
A novel 3D-printed 24-well holder enabled scalable in situ confocal imaging of
cells in Transwell® inserts. ‘In focus’ junction complexes in each z plane
were quantified by image analysis algorithms. Change in log10(mean TEER) was
used to statistically evaluate barrier integrity treatments. TGF-b reduced
barrier integrity (TEER and junction complex
‘total area’). This TEER-imaging approach is being used for allergic
inflammation target validation. It has potential use for other epithelium and
scaling for e.g. safety assessment.