Derivation of 2-month-old porcine jejunum-derived small intestinal organoids for in vitro

Background: Three-dimensional (3D) intestinal organoids have emerged as robust in vitro platforms that closely mimic native mucosal architecture, offering promising alternatives to animal experimentation.Developing 2-month-old piglet-derived small intestinal organoids is especially critical for establishing a physiologically relevant developmental model to study gut epithelial remodeling and mucosal adaptations during the post-weaning transition.Methods: To model post-weaning gut remodeling, 2-month-old piglet jejunum tissues were verified histologically, and segment-and age-dependent transcriptomes were comparatively profiled.Subsequently, small intestinal organoids were established by culturing isolated 2-month-old jejunum intestinal crypts in 3D Matrigel.Results: Histological evaluation confirmed intact intestinal crypt-villus integrity in 2-month-old tissues.Transcriptomic analysis revealed prominent epithelial marker enrichment and reduced LGR5 expression in 2-month-old jejunum relative to agematched ileum and adult jejunum.In 3D culture, 2-month-old jejunum intestinal crypts successfully formed multi-budded organoids, displaying typical differentiation patterns and robust expansion kinetics.Conclusions: In this study, we successfully derived 2-month-old porcine jejunumderived intestinal organoids in 3D culture.Accompanied by further downstream characterization, this platform integrated with organ-on-a-chip technology will serve as a valuable in vitro model for assessing nutrient absorption, ultimately contributing to enhanced swine productivity in animal biotechnology.