Organoids (‘mini-organs’) derived from adult stem cells residing in tissues can provide near-physiological in vitro ‘Reduction, Refinement, and Replacement (3Rs)’ models for genotype-to-phenotype research in animal sciences. Our aim was to assess the effective ability and reliability of pig intestinal organoids to reflect the phenotypes of the specific gut portion they are derived from. To this issue we generated organoids from the small intestine (duodenum, jejunum, ileum) and colon from cryopreserved biopsies of four Large White pigs after adapting the protocol to commercial slaughter conditions and further standardization of lab culture and phenotyping procedures. Here we report the comparison of organoids cultured in three and two dimensions (3D and 2D) vs the original animal tissue in terms of morphology, number and growth rate, cell composition and barrier tissue integrity, and highlight the main organoid vs tissue specificities.