Porcine Intestinal Apical-Out Organoid Model for Gut Function Study

Abstract

Pig models provide valuable research information on farm animals, veterinary, and biomedical sciences. Experimental pig gut models are used in studies on physiology, nutrition, and diseases. Intestinal organoids are powerful tools for investigating intestinal functions such as nutrient uptake and gut barrier function. However, organoids have a basal-out structure and need to grow in the extracellular matrix, which causes difficulties in research on the intestinal apical membrane. We established porcine intestinal organoids from jejunum tissues and developed basal-out and apical-out organoids using different sub-culture methods. Staining and quantitative real-time PCR showed the difference in axis change of the membrane and gene expression of epithelial cell marker genes. To consider the possibility of using apical-out organoids for intestinal function, studies involving fatty acid uptake and disruption of the epithelial barrier were undertaken. Fluorescence fatty acid was more readily absorbed in apical-out organoids than in basal-out organoids within the same time. To determine whether apical-out organoids form a functional barrier, a fluorescent dextran diffusion assay was performed. Hence, we successfully developed porcine intestinal organoid culture systems and showed that the porcine apical-out organoid model is ideal for the investigation of the intestinal environment. It can be used in future studies related to the intestine across various research fields.

Establishment of a chicken intestinal organoid culture system to assess deoxynivalenol-induced damage of the intestinal barrier function

“…The apical side of the intestinal epithelium is exposed to the lumen, and the associated membrane interacts with environmental stimulation and microorganisms as well as facilitating nutrient absorption [ 36 ]. In general, organoid cultures have a basal-out structure with the apical membrane facing forward to the interior, which is different from the organization of the small intestine epithelium and may affect the assessment of the nutrient uptake and barrier function in organoids compared to the actual chicken small intestinal epithelium [ 37 ]. Therefore, we developed an apical-out organoid, estimated its permeability, and compared its nutrient intake abilities with those of the basal-out organoids.…”

The World of Organoids: Gastrointestinal Disease Modelling in the Age of 3R and One Health with Specific Relevance to Dogs and Cats

“…Interestingly, intestinal apical-out organoids have been explored intensely in different animal species but not so much in mouse and human organoids over the last few years. A study using pig organoids analysed their potential to form apical-out organoids and set up functional readouts as fatty acid uptake and barrier integrity analyses [ 82 ]. There are several groups working on apical-out organoids for disease modelling in different contexts.…”

Polarity reversal of canine intestinal organoids reduces proliferation and increases cell death

“…This method has now been adopted by many other labs and expanded to different animal species, for example, pigs, 13 chickens, 14 cows 15 and dogs, 16 and also organoids derived from human iPSCs and embryonic stem cells 17,18 . Remarkably, regardless of the species from which the organoids were derived, the majority of these studies utilised their apical‐out organoids within the initial 7 days following the induction of polarity reversal 13,16,17,19–21 . Experiences from our lab show, that canine intestinal apical‐out organoids produce a lot of cell debris within the culture well, which is most probably due to dying cells being extruded from the epithelial layer of cells directly into the medium.…”

“… 17 , 18 Remarkably, regardless of the species from which the organoids were derived, the majority of these studies utilised their apical‐out organoids within the initial 7 days following the induction of polarity reversal. 13 , 16 , 17 , 19 , 20 , 21 Experiences from our lab show, that canine intestinal apical‐out organoids produce a lot of cell debris within the culture well, which is most probably due to dying cells being extruded from the epithelial layer of cells directly into the medium. This phenomenon increases until apical‐out organoids slowly start to die off completely after approximately 1 week of culture.…”