Organoids as a Model for Intestinal Ion Transport Physiology

The advent of intestinal organoid culture in 2009 was a fortuitous development in the search for a valid marker of intestinal stem cells, and provided proof of murine intestinal stem cell regenerative potential. Intestinal organoid culture was preceded by key discoveries of the Wnt/β-catenin signaling pathway and the development of 3D culture matrices. The latter, involving a laminin-rich gel to provide an artificial basement membrane, was instrumental to primary intestinal epithelial culture by preventing anoikis, an immediate apoptotic event when intestinal epithelial cells detach from the basement membrane. One of the first physiological studies using 3D murine “mini-gut” structures showed cystic fibrosis transmembrane conductance regulator (CFTR) expression and anion channel activity in the crypt-like structures projecting from the epithelial-lined central cavity. Detailed investigations of ion transport physiology using human intestinal organoids, both primary and iPSC-derived, found close similarities to existing knowledge of ion transport physiology and included the development of the forskolin-induced swelling assay (FIS). The FIS assay using organoids cultured from rectal biopsies of cystic fibrosis patients provided an avenue for personalized medicine to test small-molecule modulators on different CFTR mutations. More recent research has led to the development of 2D primary intestinal epithelial monolayers, which provide easy access to the apical, lumen-facing membrane and the opportunity for traditional ion transport studies with Ussing chambers. Human 2D primary intestinal monolayers also demonstrate the dominance of CFTR in anion secretion and provide a quantitative evaluation of its chloride and bicarbonate secretory conductances. These aspects of ion transport physiology using 2D and 3D intestinal cultures are discussed along with the relative advantages and disadvantages of each culture method with respect to technical aspects and recapitulation of native intestinal epithelium

The use of murine-derived fundic organoids in studies of gastric physiology

Key points: An in vitro approach to study gastric development is primary mouse-derived epithelium cultured as three-dimensional spheroids known as organoids. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Organoids maintained in co-culture with immortalized stomach mesenchymal cells express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. We report the use of these models for studies of epithelial cell biology and cell damage and repair. Studies of gastric function and disease have been limited by the lack of extended primary cultures of the epithelium. An in vitro approach to study gastric development is primary mouse-derived antral epithelium cultured as three-dimensional spheroids known as organoids. There have been no reports on the use of organoids for gastric function. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Both models were generated from single glands dissociated from whole fundic tissue and grown in basement membrane matrix (Matrigel) and organoid growth medium. Model 1 enriches for a stem cell-like niche via simple passage of the organoids. Maintained in Matrigel and growth medium, proliferating organoids expressed high levels of stem cell markers CD44 and Lgr5. Model 2 is a system of gastric organoids co-cultured with immortalized stomach mesenchymal cells (ISMCs). Organoids maintained in co-culture with ISMCs express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. Thus, we report the use of these models for studies of epithelial cell biology and cell damage and repair