A Human 2D Primary Organoid-Derived Epithelial Monolayer Model to Study Host-Pathogen Interaction in the Small Intestine

Abstract

Gut organoids are stem cell derived 3D models of the intestinal epithelium that are useful for studying interactions between enteric pathogens and their host. While the organoid model has been used for both bacterial and viral infections, this is a closed system with the luminal side being inaccessible without microinjection or disruption of the organoid polarization. In order to overcome this and simplify their applicability for transepithelial studies, permeable membrane based monolayer approaches are needed. In this paper, we demonstrate a method for generating a monolayer model of the human fetal intestinal polarized epithelium that is fully characterized and validated. Proximal and distal small intestinal organoids were used to generate 2D monolayer cultures, which were characterized with respect to epithelial cell types, polarization, barrier function, and gene expression. In addition, viral replication and bacterial translocation after apical infection with enteric pathogens Enterovirus A71 and Listeria monocytogenes were evaluated, with subsequent monitoring of the pro-inflammatory host response. This human 2D fetal intestinal monolayer model will be a valuable tool to study host-pathogen interactions and potentially reduce the use of animals in research.

A Perspective on Organoids for Virology Research

“…These round structures are difficult to infect with viruses: receptors needed for infection are mostly located inside the organoid. As with HAE cultures, round gut organoids can be transformed to an open organoid model where cells grow on a Transwell ® , so that they can be reached from the upper and lower sides to establish infections [ 6 , 26 , 42 , 43 ]. This model system is ideal for infectious-disease studies and to test antimicrobial drugs.…”

Enteropathogenic Infections: Organoids Go Bacterial

“…Previous reports indicated that L. monocytogenes preferably traverses the intestinal epithelium both through goblet cells and M cells at the Peyer's patch level [ 71 , 72 ]. Recently introduced by Roodsant et al as an equivalent novel organoid culture model, human fetal tissue-derived intestinal organoids were plated as a monolayer and apically inoculated with L. monocytogenes which predominantly colocalized with MUC 2-positive goblet cells [ 73 ]. Furthermore, it was noted that the fluorescent staining signal for actin became weaker in the apical region of infected cells [ 73 ].…”

“…Recently introduced by Roodsant et al as an equivalent novel organoid culture model, human fetal tissue-derived intestinal organoids were plated as a monolayer and apically inoculated with L. monocytogenes which predominantly colocalized with MUC 2-positive goblet cells [ 73 ]. Furthermore, it was noted that the fluorescent staining signal for actin became weaker in the apical region of infected cells [ 73 ]. This finding might be linked to the property of L. monocytogenes to rearrange the host cell's actin into so-called “comet tails” to facilitate intracellular mobility, as previously reported by Co et al in human small intestinal organoids [ 65 ].…”

A Novel Strategy to Study the Invasive Capability of Adherent-Invasive Escherichia coli by Using Human Primary Organoid-Derived Epithelial Monolayers

“…Such an approach could lead to the discovery of new disease biomarkers and new therapeutic targets. To our knowledge, there are few publicly available reports that analyze the interaction between enteric pathogens and human isolated IECs (23,(37)(38)(39)(40)(41). More recently, Sayed et al published a study in which AIEC infection of organoid-derived 2D cultures is applied to explore host engulfment in IBD.…”