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.
Highlights
The intestinal epithelium consists of a single layer of polarized cells that separates the intestinal content from the rest of the body
The integrity of human fetal organoid-derived epithelial monolayers in culture was evaluated by performing longitudinal measurements of transepithelial electrical resistance (TEER) for 28 days, a widely accepted quantitative technique to measure the integrity of tight junctions in cell culture models of epithelial monolayers (Srinivasan et al, 2015)
Based on the observed stabilization of trans-epithelial electrical resistance (TEER) and smallest biological variation for both proximal and distal cultures, day 14 of culture was identified as time point with the optimal epithelial integrity for monolayers derived from fetal intestinal organoids
Summary
The intestinal epithelium consists of a single layer of polarized cells that separates the intestinal content from the rest of the body. For instance caused by microbial signals from pathogenic bacteria, can increase the susceptibility to infections and result in various intestinal and extra-intestinal diseases (Rahman et al, 2016; Ni et al, 2017; Manfredo Vieira et al, 2018; Rinninella et al, 2019) These host-pathogen interaction studies were performed in vitro using immortalized cancer cell lines which bear various mutations, resulting in aberrant proliferation and differentiation in comparison to in vivo intestinal epithelium (Sun et al, 2008; Maidji et al, 2017; von Martels et al, 2017; Calatayud et al, 2018). Another disadvantage of these models is the lack of diversity in specialized epithelial cell types, for example the Caco-2 cell line consists predominantly of enterocytes (Sun et al, 2008)
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