4.7 Article

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

Journal

POULTRY SCIENCE
Volume 101, Issue 3, Pages -

Publisher

ELSEVIER
DOI: 10.1016/j.psj.2021.101642

Keywords

three-dimensional cell culture; organoid; crypt; chicken intestines

Funding

  1. NSF ADVANCE Institutional Transformation Award [1008385]
  2. College of Agriculture and Life Science at Texas AM University
  3. USDA NIFA Multistate Hatch [TEX07712]
  4. USDA-ARS [505584-97090]
  5. Allen Endowed Chair in Nutrition & Chronic Disease Prevention (RSC)
  6. National Institutes of Health [R35-CA197707]
  7. Direct For Education and Human Resources
  8. Division Of Human Resource Development [1008385] Funding Source: National Science Foundation

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This study successfully generated crypt-derived organoids from chicken intestines and optimized conditions for their growth and cryopreservation. The developed model will contribute to understanding host-pathogen interactions and discovering pathogen intervention strategies in poultry.
Intestinal organoids (IO), known as mini-guts, derived from intestinal crypts, are self-organizing three-dimensional (3D) multicellular ex vivo models that recapitulate intestine epithelial structure and function and 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 model for poultry research is lacking. Therefore, 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 the small intestines of birds at embryonic d-19 and ceca from layer and broiler chickens with ages ranging from d 1 to 20 wk, 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 3D spheres like structures that were cultured for up to 3 wk. Organoids were formed on d one, budding appeared on d 3, and robust budding was observed on d 7 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 RTPCR 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 epithelial cells, stem cells, enteroendocrine cells, Paneth cells, and goblet cells. Furthermore, OGM supplemented with both valproic acid and CHIR99021, a glycogen synthase kinase 3b inhibitor and a histone deacetylase inhibitor, increased the size of the avian IO (P < 0.001). To the best of our knowledge, this is the first comprehensive report for establishing long-term, organoid culture models from small intestines and ceca of layer and broiler chickens. This model will facilitate elucidation of the mechanisms impacting host-pathogen interactions, eventually leading to the discovery of pathogen intervention strategies in poultry.

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