Development of Arrayed Colonic Organoids for Screening of Secretagogues Associated with Enterotoxins

Abstract

Enterotoxins increase intestinal fluid secretion through modulation of ion channels as well as activation of the enteric nervous and immune systems. Colonic organoids, also known as colonoids, are functionally and phenotypically similar to in vivo colonic epithelium and have been used to study intestinal ion transport and subsequent water flux in physiology and disease models. In conventional cultures, organoids exist as spheroids embedded within a hydrogel patty of extracellular matrix, and they form at multiple depths, impairing efficient imaging necessary to capture data from statistically relevant sample sizes. To overcome these limitations, an analytical platform with colonic organoids localized to the planar surface of a hydrogel layer was developed. The arrays of densely packed colonoids (140 μm average diameter, 4 colonoids/mm) were generated in a 96-well plate, enabling assay of the response of hundreds of organoids so that organoid subpopulations with distinct behaviors were identifiable. Organoid cell types, monolayer polarity, and growth were similar to those embedded in hydrogel. An automated imaging and analysis platform efficiently tracked over time swelling due to forskolin and fluid movement across the cell monolayer stimulated by cholera toxin. The platform was used to screen compounds associated with the enteric nervous and immune systems for their effect on fluid movement across epithelial cells. Prostaglandin E2 promoted increased water flux in a subset of organoids that resulted in organoid swelling, confirming a role for this inflammatory mediator in diarrheal conditions but also illustrating organoid differences in response to an identical stimulus. By allowing sampling of a large number of organoids, the arrayed organoid platform permits identification of organoid subpopulations intermixed within a larger group of nonresponding organoids. This technique will enable automated, large-scale screening of the impact of drugs, toxins, and other compounds on colonic physiology.

A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology

“…Although 16S rRNA gene amplicon sequencing indicates the presence and relative abundance of bacterial taxa, it does not access their viability. To validate whether nonsporulating obligately anaerobe species of the Bifidobacterium genera could survive and grow within the colonoid lumen, a monoculture of B adolescentis , a highly oxygen-intolerant species that is an early colonizer of the infant gut, 59 was microinjected into colonoids ( Figure 5 H ). Survival and growth of B adolescentis in the colonoid lumen was evaluated at 6, 12, 24, 48, or 60 hours after microinjection.…”

Intestinal Organoid Culture in Polymer Film‐Based Microwell Arrays

“…This feature also helps to simplify organoid tracking throughout the experiment and promotes high-content image acquisition. Furthermore, microwell culturing of organoids facilitates additional experimental manipulations, such as microinjections [40,52,53] and co-culturing. Finally, this microwell-based organoid culture platform could be combined with other systems, such as microfluidics for screening applications, where the exchange of tested substances and soluble factors can be performed in a more controlled way, yet without necessarily creating increased shear stress for the organoids, which are protected inside the microwells.…”

Germ‐free and microbiota‐associated mice yield small intestinal epithelial organoids with equivalent and robust transcriptome/proteome expression phenotypes

“…These stem cells divide and give rise to epithelial cell precursors, which differentiate into paneth cells, enteroendocrine cells, goblet cells and enterocytes, hence recapitulating much of the complexity of co‐existing cell types in the gut mucosa (Foulke‐Abel et al, ; Sato et al, ). For these reasons, organoids have since their conception become a widely used and realistic model to study the role of intestinal epithelial cells in, for example, gut physiology (Almeqdadi, Mana, Roper, & Yilmaz, ; Gunasekara et al, ; Williamson et al, ), cancer biology (Drost et al, ; Tuveson & Clevers, ), pharmacology (Takahashi, ; Walsh, Cook, Sanders, Arteaga, & Skala, ) and infectious disease (Co et al, ; Foulke‐Abel et al, ; Hausmann & Hardt, ; Sun, ; Zhang, Wu, Xia, & Sun, ). It appears conceivable that organoids over time will replace traditional cell lines as the main tissue culture model of choice for mechanistic studies.…”