Effect of substrate stiffness on human intestinal enteroids' infectivity by enteroaggregative Escherichia coli

Human intestinal enteroids (HIE) models have contributed significantly to our understanding of diarrheal diseases and other intestinal infections, but their routine culture conditions fail to mimic the mechanical environment of the native intestinal wall. Because the mechanical characteristics of the intestine significantly alter how pathogens interact with the intestinal epithelium, we used different concentrations of polyethylene glycol (PEG) to generate soft (~2 kPa), medium (~10 kPa), and stiff (~100 kPa) hydrogel biomaterial scaffolds. The height of HIEs cultured in monolayers atop these hydrogels was 18 µm whereas HIEs grown on rigid tissue culture surfaces (with stiffness in the GPa range) were 10 µm. Substrate stiffness also influenced the amount of enteroaggregative E. coli (EAEC strain 042) adhered to the HIEs. We quantified a striking difference in adherence pattern; on the medium and soft gels, the bacteria formed clusters of > 100 and even > 1000 on both duodenal and jejunal HIEs (such as would be found in biofilms), but did not on glass slides and stiff hydrogels. All hydrogel cultured HIEs showed significant enrichment for gene and signaling pathways related to epithelial differentiation, cell junctions and adhesions, extracellular matrix, mucins, and cell signaling compared to the HIEs cultured on rigid tissue culture surfaces. Collectively, these results indicate that the HIE monolayers cultured on the hydrogels are primed for a robust engagement with their mechanical environment, and that the soft hydrogels promote the formation of larger EAEC aggregates, likely through an indirect differential effect on mucus. STATEMENT OF SIGNIFICANCE: Enteroids are a form of in vitro experimental mini-guts created from intestinal stem cells. Enteroids are usually cultured in 3D within Matrigel atop rigid glass or plastic substrates, which fail to mimic the native intestinal mechanical environment. Because intestinal mechanics significantly alter how pathogens interact with the intestinal epithelium, we grew human intestinal enteroids in 2D atop polyethylene glycol (PEG) hydrogel scaffolds that were soft, medium, or stiff. Compared with enteroids grown in 2D atop glass or plastic, the enteroids grown on hydrogels were taller and more enriched in mechanobiology-related gene signaling pathways. Additionally, enteroids on the softest hydrogels supported adhesion of large aggregates of enteroaggregative E. coli. Thus, this platform offers a more biomimetic model for studying enteric diseases.