Development of sheep duodenum intestinal organoids and implementation of high-throughput screening platform for veterinary applications

19 páginas, 8 figuras, 2 tablas.

New therapeutic molecules for farm animals are needed to address worldwide problems in the food industry, like the rise of resistance among ruminant parasites and pathogenic microbes. Since in vivo testing would involve an excessive number of animals, with consequent ethical and economic issues, the generation of sheep intestinal organoids represents a promising close-to-reality in vitro model for veterinary drug development; however, the characterization and application of such organoids remain limited. In this study, ovine intestinal organoids were generated from adult LGR5+ stem cells from the intestinal crypts of freshly slaughtered lambs, and developed in an in vitro culture system. Morphological analysis via brightfield microscopy and immunocytochemical staining revealed a pseudostratified epithelium with multiple cell types, and distinct apical–basal polarity, while RNA sequencing validated the preservation of the physiological characteristics of the original organ. The development and characterization of a robust and reproducible protocol for culturing sheep duodenum intestinal organoids in a high-throughput screening (HTS) compatible format demonstrated reliability in HTS applications, with Z’-factor tests indicating robust assay performance. Dose–response studies using pre-identified compounds showed comparable pharmacodynamic profiles between mouse and sheep organoids. These findings establish sheep intestinal organoids as an innovative tool for veterinary pharmacology and toxicology, offering a cost-effective and sustainable platform to address challenges such as drug resistance and improve livestock health.

This research was part of projects PID2020-119035RB-I00 and PID2021-126489OB-I00, funded by Ministerio Ciencia, Innovación y Universidades (MICIU)/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”. This research also received funds from University of León and Fundación General de la Universidad de León y de la Empresa (FGULEM) through the ULE-PoC (PRUEBAS DE CONCEPTO DE LA UNIVERSIDAD DE LEÓN, Plan TCUE 2023-2027) programme. Grant PRE2021-096909 was funded by MICIU/AEI/10.13039/501100011033 and by “ESF Investing in your future”.

Peer reviewed