Establishing a human in vitro 3D gut-on-a-chip model to study enterohemorrhagic Escherichia coli infection

Introduction

Enterohemorrhagic Escherichia coli (EHEC) are pathogenic bacteria that can cause severe intestinal infections and the hemolytic uremic syndrome. To study EHEC infections, cell culture systems or animal models can be applied. As none of the currently available animal models fully reflects EHEC pathogenesis in humans, human 3D cell culture systems mimicking in vivo conditions are a promising technology for monitoring EHEC infection.

Goals

We intend to establish and characterize an easy-to-use 3D cell culture model comprising four different cell types that mimics the human gut and is suitable for studying EHEC infections.

Materials & Methods

Caco-2 alone or together with HT29-MTX intestinal epithelial cells were seeded against extracellular matrix (ECM) in an OrganoPlate® 3-lane 40 (Mimetas). Cells were cultivated under flow conditions until leak-tight intestinal tubules were formed. Infection experiments with bacterial culture supernatant for 24 h or bacteria for 3 h were carried out using the 2011 outbreak strain EHEC O104:H4. Prior and after infection, barrier integrity was assessed by monitoring the translocation of fluorophore-labeled dextrans from tubule lumen to ECM channel.

Results

Leak-tight intestinal tubules were formed after six days of cultivation. Infection of tubules with culture supernatant from EHEC O104:H4 wild type, but not from its Shiga toxin 2 (Stx2) phage-cured derivative, induced epithelial barrier disruption, reflecting the known cytotoxic effect of Stx2. Additionally, infection experiments with bacteria revealed that an EHEC O104:H4 rpoS-mutant with similar Stx2 levels, but enhanced fimbriae and flagellin production compared to the wild type, caused a more severe tubule barrier disruption, indicating synergistic effects of Stx2 and these virulence factors.

Summary

So far, we established an easy-to-handle 3D bi-culture model suitable for studying EHEC infections in vitro. To fully picture host defense and EHEC disease pathology, we are currently implementing macrophages and neutrophils into the 3D cell culture system.