culture-piglet-intestinal-3d-organoids-from-cryopreserved-epithelial

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

Université de Toulouse, INRAE, ENVT

Intestinal organoids are self-organized 3-dimensional (3D) structures formed by a single layer of polarized epithelial cells. This innovative in vitro model is highly relevant to study physiology of the intestinal epithelium and its role in nutrition and barrier function. However, this model has never been developed in rabbits, while it would have potential applications for biomedical and veterinary research. Here, we cultured rabbit caecum organoids with either pharmacological inhibitors (2Ki medium) or L-WRN cells conditioned medium (L-WRN CM) to reconstitute the intestinal stem cell niche in vitro. Large spherical organoids were obtained with the 2Ki medium and this morphology was associated with a high level of proliferation and stem cells markers gene expression. In contrast, organoids cultured with L-WRN CM had a smaller diameter; a greater cell height and part of them were not spherical. When the L-WRN CM was used at low concentration (5%) for two days, the gene expression of stem cells and proliferation markers were very low, while absorptive and secretory cells markers and antimicrobial peptides were elevated. Epithelial cells within organoids were polarized in 3D cultures with 2Ki medium or L-WRN CM (apical side towards the lumen). We cultured dissociated organoid cells in 2D monolayers, which allowed accessibility to the apical compartment. Under these conditions, actin stress fibers were observed with the 2Ki medium, while perijonctionnal localization of actin was observed with the L-WRN CM suggesting, in 2D cultures as well, a higher differentiation level in the presence of L-WRN CM. In conclusion, rabbit caecum organoids cultured with the 2Ki medium were more proliferative and less differentiated than organoids cultured with L-WRN CM. We propose that organoids cultured with the 2Ki medium could be used to rapidly generate in vitro a large number of rabbit intestinal epithelial stem cells while organoids cultured with the L-WRN CM used at low concentration represent a suitable model to study differentiated rabbit epithelium.

Culture of rabbit caecum organoids by reconstituting the intestinal stem cell niche in vitro with pharmacological inhibitors or L-WRN conditioned medium.

In livestock species, the monolayer of epithelial cells covering the digestive mucosa plays an essential role for nutrition and gut barrier function. However, research on farm animal intestinal epithelium has been hampered by the lack of appropriate in vitro models. Over the past decade, methods to culture livestock intestinal organoids have been developed in pig, bovine, rabbit, horse, sheep and chicken. Gut organoids from farm animals are obtained by seeding tissue-derived intestinal epithelial stem cells in a 3-dimensional culture environment reproducing in vitro the stem cell niche. These organoids can be generated rapidly within days and are formed by a monolayer of polarized epithelial cells containing the diverse differentiated epithelial progeny, recapitulating the original structure and function of the native epithelium. The phenotype of intestinal organoids is stable in long-term culture and reflects characteristics of the digestive segment of origin. Farm animal intestinal organoids can be amplified in vitro, cryopreserved and used for multiple experiments, allowing an efficient reduction of the use of live animals for experimentation. Most of the studies using livestock intestinal organoids were used to investigate host-microbe interactions at the epithelial surface, mainly focused on enteric infections with viruses, bacteria or parasites. Numerous other applications of farm animal intestinal organoids include studies on nutrient absorption, genome editing and bioactive compounds screening relevant for agricultural, veterinary and biomedical sciences. Further improvements of the methods used to culture intestinal organoids from farm animals are required to replicate more closely the intestinal tissue complexity, including the presence of non-epithelial cell types and of the gut microbiota. Harmonization of the methods used to culture livestock intestinal organoids will also be required to increase the reproducibility of the results obtained in these models. In this review, we summarize the methods used to generate and cryopreserve intestinal organoids in farm animals, present their phenotypes and discuss current and future applications of this innovative culture system of the digestive epithelium.

Intestinal organoids in farm animals.

Weaning is a challenging period for gut health in piglets. Previous studies showed that dietary supplementations with either amino acids or polyphenols promote piglet growth and intestinal functions, when administered separately. Thus, we hypothesized that a combination of amino acids and polyphenols could facilitate the weaning transition. Piglets received during the first two weeks after weaning a diet supplemented or not with a mix of a low dose (0.1%) of functional amino acids (L-arginine, L-leucine, L-valine, L-isoleucine, L-cystine) and 100 ppm of a polyphenol-rich extract from grape seeds and skins. The mix of amino acids and polyphenols improved growth and feed efficiency. These beneficial effects were associated with a lower microbiota diversity and a bloom of Lactobacillaceae in the jejunum content while the abundance of Proteobacteria was reduced in the caecum content. The mix of amino acids and polyphenols also increased the production by the caecum microbiota of short-chain fatty acids (butyrate, propionate) and of metabolites derived from amino acids (branched-chain fatty acids, valerate, putrescine) and from polyphenols (3-phenylpropionate). Experiments in piglet jejunum organoids revealed that the mix of amino acids and polyphenols upregulated the gene expression of epithelial differentiation markers while it reduced the gene expression of proliferation and innate immunity markers. In conclusion, the supplementation of a mix of amino acids and polyphenols is a promising nutritional strategy to manage gut health in piglets through the modulation of the gut microbiota and of the epithelial barrier.

A mix of functional amino acids and grape polyphenols promotes the growth of piglets, modulates the gut microbiota in vivo and regulates epithelial homeostasis in intestinal organoids.

Martin Beaumont

GenPhySE

Université de Toulouse, INRAE, ENVT

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Martin Beaumont

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}GenPhySE

Université de Toulouse, INRAE, ENVT

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

GenPhySE