From crypts to enteroids: Establishment and characterization of avian intestinal organoids

Authors: ZHAO, DAN - Texas A&M University; FARNELL, MORGAN - Texas A&M University; Kogut, Michael - Mike; Genovese, Kenneth - Ken; CHAPKIN, ROBERT - Texas A&M University; DAVIDSON, LAURIE - Texas A&M University; BERGHMAN, LUC - Texas A&M University; FARNELL, YUHUA - Texas A&M University

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/17/2021
Publication Date: 3/1/2022
Citation: Zhao, D., Farnell, M.B., Kogut, M.H., Genovese, K.J., Chapkin, R.S., Davidson, L.A., Berghman, L.R., Farnell, Y.Z. 2022. From crypts to enteroids: Establishment and characterization of avian intestinal organoids. Poultry Science. 101(3). Article 101642. https://doi.org/10.1016/j.psj.2021.101642.
DOI: https://doi.org/10.1016/j.psj.2021.101642

Interpretive Summary: The intestines of young chickens were collected, treated, and cultured to establish an intestinal organoid culture which can be used for studying intestinal functions in birds. The organoids were characterized using multiple molecular methods as well as live cell imaging. This is an especially important step to understanding the relationship and responses of the chicken gastrointestinal tract with various foodborne pathogens including Salmonella and Campylobacter.

Technical Abstract: Intestinal organoids (IO), known as "mini-guts", derived from intestinal crypts, are self-organizing three-dimensional (3-D) multicellular ex vivo models that recapitulate intestine epithelial structure and function. These have been widely used for studying intestinal physiology, pathophysiology, molecular mechanisms of host-pathogen interactions, and intestinal disease in mammals. However, studies on avian IO are limited and the development of long-term cultures of IO models for poultry research is lacking. The objectives of this study were to generate crypt-derived organoids from chicken intestines and to optimize conditions for cell growth and enrichments, passages, and cryopreservation. Crypts were collected from small intestines of birds at embryonic day-19 and ceca from layer and broiler chickens with ages ranging from day one to twenty weeks, embedded in a basement membrane matrix, and cultured with organoid growth media (OGM) prepared in house. The crypt-derived organoids were successfully grown and propagated to form 3-D sphere like structures that were cultured for up to three weeks. Organoids were formed on day one, budding appeared on day three, and robust budding was observed on day seven and beyond. For cryopreservation, dissociated organoids were resuspended in a freezing medium. The characteristics of IO upon extended passages and freeze-thaw cycles were analyzed using reverse transcription (RT)-PCR, immunoblotting, and live cell imaging. Immunoblotting and RT-PCR using E-cadherin (the marker for epithelial cells), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5, the marker for stem cells), chromogranin A (the marker for enteroendocrine cells), lysozyme (the marker for Paneth cells), and mucin (the biomarker for goblet cells) confirmed that IO were composed of heterogeneous cell populations, including enterocytes, stem cells, enteroendocrine cells, Paneth cells, and goblet cells. Furthermore, OGM supplemented with both Valproic acid and CHIR99021, a glycogen synthase kinase 3 beta inhibitor and a histone deacetylase inhibitor, increased the size of the avian IO (P < 0.001). To the best of our knowledge, this work was the first comprehensive report for establishing long-term, organoid culture models from small intestines and ceca of layer and broiler chickens, which will help us better understand host-pathogen interactions, eventually leading to the discovery of pathogen intervention strategies in poultry.