Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis

Abstract

There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining IgA transcytosis across Transwells. IgA transcytosis required induction of polymeric immunoglobulin receptor (pIgR) expression, which could be stimulated by a combination of LPS and inhibition of γ-secretase. In agreement with previous studies using immortalized cell lines, we found that TNFα, IL-1β, IL-17 and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that amongst these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. IFNγ however did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology.

Expression of Human Decay-Accelerating Factor on Intestinal Epithelium of Transgenic Mice Does Not Facilitate Infection by the Enteral Route

“…Because a complex set of DAF-dependent intracellular signals and cytoskeletal rearrangements is required for CVB3 infection of polarized human intestinal epithelium (10, 36), we wanted to be sure that human DAF functions to permit infection of murine cells. To test this, we made use of recent technical advances in the culture and maintenance of nontransformed primary epithelial cells (27,37,38). We isolated intestinal stem cells from the duodenum of a DAF transgenic mouse and from a wild-type littermate and used these to produce polarized epithelial cell monolayers.…”

Using 3D Organoid Cultures to Model Intestinal Physiology and Colorectal Cancer

“…Others have instead opted to grow enteroids or colonoids as monolayers on transwells, which disrupts 3D architecture but preserves differentiation, allows polarization and exposes the apical surface. While this model has proven useful for studies of barrier function, transcytosis, and cell polarity, it also allows ready infection and detection of attaching and effacing lesions associated with infection [55, 56]. Conversely, intraluminal injection methods have been used to directly inoculate into the enteroid lumen, although this is a much more time consuming model and not amenable to high-throughput applications [57–59].…”

Determining Microbial Products and Identifying Molecular Targets in the Human Microbiome

“…Testing the effects of microbial products on host cell function requires three components: a host cell system to perturb, a microbially-derived perturbation, and a readout of host cell state. The first component may be a model organism, gnotobiotic animal, cell culture, organoid (Sato and Clevers, 2013), or a structured culture such as a transwell (Moon et al, 2014) or gut-on-chip (Huh et al, 2013). The second component may include live or inactivated microbial cells, whole lysate, media, or an isolated, purified, or synthesized product.…”