Intestinal organoid-based 2D monolayers mimic physiological and pathophysiological properties of the pig intestine

Abstract

Gastrointestinal infectious diseases remain an important issue for human and animal health. Investigations on gastrointestinal infectious diseases are classically performed in laboratory animals leading to the problem that species-specific models are scarcely available, especially when it comes to farm animals. The 3R principles of Russel and Burch were achieved using intestinal organoids of porcine jejunum. These organoids seem to be a promising tool to generate species-specific in vitro models of intestinal epithelium. 3D Organoids were grown in an extracellular matrix and characterized by qPCR. Organoids were also seeded on permeable filter supports in order to generate 2D epithelial monolayers. The organoid-based 2D monolayers were characterized morphologically and were investigated regarding their potential to study physiological transport properties and pathophysiological processes. They showed a monolayer structure containing different cell types. Moreover, their functional activity was demonstrated by their increasing transepithelial electrical resistance over 18 days and by an active glucose transport and chloride secretion. Furthermore, the organoid-based 2D monolayers were also confronted with cholera toxin derived from Vibrio cholerae as a proof of concept. Incubation with cholera toxin led to an increase of short-circuit current indicating an enhanced epithelial chloride secretion, which is a typical characteristic of cholera infections. Taken this together, our model allows the investigation of physiological and pathophysiological mechanisms focusing on the small intestine of pigs. This is in line with the 3R principle and allows the reduction of classical animal experiments.

Porcine Intestinal Apical-Out Organoid Model for Gut Function Study

“…Glutamate enhances pig organoid development and epithelial cell proliferation via mTORC1 activation, which includes IR, IRS, PI3K, and Akt pathways [33]. Additionally, pig organoids showed a pathophysiological response to cholera toxin, a bacteria-derived toxin [15]. Although an organoid system has various advantages, gut organoids, especially basal-out organoids, have a critical limitation for gut functional studies.…”

“…However, the basal-out organoids have limited access to luminal materials such as nutrients and microbes. Thus, researchers often used 2D monolayer organoid culture models to overcome the structural limitations of basal-out organoids [15]. Monolayer pig organoids showed cellular functional transport characteristics, such as SGLT1 and CFTR.…”

“…In epithelial tight junction and permeability tests, porcine ileum organoids were used as a form of two-dimensional (2D) monolayer [14]. In this system, nutrient uptake (glucose absorption) and reactions to bacterial enterotoxins were examined [15]. Another example is a new organoid cultivation technique that was developed using suspension culture [16].…”

Farm and Companion Animal Organoid Models in Translational Research: A Powerful Tool to Bridge the Gap Between Mice and Humans

“…More recently, organoid-derived 2D monolayer culture models have become increasingly described in multiple animal species including pigs ( 23 , 46 , 47 , 49 – 51 , 53 , 54 , 58 , 62 , 64 ), rabbits ( 97 , 98 ), cattle ( 72 ), and dogs ( 11 ). This culture system was developed to get over the limitation of 3D culture system which arose when intestinal organoids are to be used in studies.…”

Porcine Intestinal Organoids: Overview of the State of the Art

“…A similar phenotype was reported for PIOs treated with deoxynivalenol, a toxin produced by mycobacteria [ 63 ]. Bacteria-derived cholera toxin treatment of PIOs causes typical signs of cholera toxin poisoning, which is characterized by increased short-circuit currents and increased epithelial chloride levels [ 64 ]. In addition to being a model for in vitro studies of pathogenic bacteria, the intestinal organoid is also a model for in vitro studies of probiotic bacteria.…”