{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T10:57:48Z","timestamp":1774349868264,"version":"3.50.1"},"posted":{"date-parts":[[2026,3,20]]},"group-title":"Cell Biology","reference-count":30,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T00:00:00Z","timestamp":1773964800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia (FCT), Portugal","award":["2021.06827.BD"],"award-info":[{"award-number":["2021.06827.BD"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,20]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Intestinal organoids are three-dimensional\n                  <jats:italic>in vitro<\/jats:italic>\n                  structures derived from stem cells and serve as a valuable model for studying intestinal biology and pathophysiology. This study optimized the isolation, expansion, and differentiation of canine intestinal organoids from duodenum and colon. Organoids were generated from canine intestinal crypts and cultured in Matrigel with a growth factor cocktail. The impact of prostaglandin E\n                  <jats:sub>2<\/jats:sub>\n                  (PGE\n                  <jats:sub>2<\/jats:sub>\n                  ) concentration on organoid growth was evaluated, and a two-phase differentiation protocol\u2014comprising patterning and differentiation media\u2014was implemented, including interleukin (IL)-22 in the duodenal differentiation phase. Organoids cultured with 100 nM PGE\n                  <jats:sub>2<\/jats:sub>\n                  exhibited increased crypt budding and organoid-forming efficiency, indicative of enhanced stem cell proliferation. Differentiated organoids expressed key intestinal markers (VIL1, SI, CHGA, MUC2), and forskolin-induced swelling demonstrated functional Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) activity. Although the sample size (n=2) limits generalizability, this optimized protocol provides a relevant\n                  <jats:italic>in vitro<\/jats:italic>\n                  model for studying canine intestinal function. The model can be used in future research for disease modelling and translational applications, supporting downstream studies in gastrointestinal disease, drug permeability, and comparative One Health research.\n                <\/jats:p>","DOI":"10.64898\/2026.03.19.712113","type":"posted-content","created":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T09:20:23Z","timestamp":1773998423000},"source":"Crossref","is-referenced-by-count":0,"title":["Optimization of isolation, expansion, and differentiation of canine intestinal organoids"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7382-7664","authenticated-orcid":false,"given":"In\u00eas E.","family":"Dias","sequence":"first","affiliation":[{"name":"Centre for the Research and Technology of Agro-environmental and Biological Sciences (CITAB), Inov4Agro \u2013 Institute for Innovation, Capacity Building and Sustainability of Agri-Food Production, Universidade de Tr\u00e1sos-Montes e Alto Douro (UTAD), Quinta de Prados, Vila Real 5000-801, Portugal"},{"name":"Vasco da Gama Research Center (CIVG), Vasco da Gama University School (EUVG), Campus Universit\u00e1rio, Av. Jos\u00e9 R. Sousa Fernandes, Lordem\u00e3o, Coimbra 3020-210, Portugal"}]},{"given":"Arabela","family":"Ritchie","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht 3584, The Netherlands"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-5281-8982","authenticated-orcid":false,"given":"Maarten","family":"Delemarre","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht 3584, The Netherlands"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8109-7657","authenticated-orcid":false,"given":"Kerstin","family":"Schneeberger","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht 3584, The Netherlands"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8796-5037","authenticated-orcid":false,"given":"Carlos A.","family":"Viegas","sequence":"additional","affiliation":[{"name":"Department of Veterinary Sciences, School of Agricultural and Veterinary Sciences (ECAV), UTAD, Quinta de Prados, Vila Real 5000-801, Portugal"},{"name":"Centre for Animal Sciences and Veterinary Studies (CECAV), AL4AnimalS\u2014Associate Laboratory for Animal and Veterinary Sciences, UTAD, Quinta de Prados, Vila Real 5000-801, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5049-4473","authenticated-orcid":false,"given":"Isabel R.","family":"Dias","sequence":"additional","affiliation":[{"name":"Department of Veterinary Sciences, School of Agricultural and Veterinary Sciences (ECAV), UTAD, Quinta de Prados, Vila Real 5000-801, Portugal"},{"name":"Centre for Animal Sciences and Veterinary Studies (CECAV), AL4AnimalS\u2014Associate Laboratory for Animal and Veterinary Sciences, UTAD, Quinta de Prados, Vila Real 5000-801, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8916-4276","authenticated-orcid":false,"given":"Pedro P.","family":"Carvalho","sequence":"additional","affiliation":[{"name":"Vasco da Gama Research Center (CIVG), Vasco da Gama University School (EUVG), Campus Universit\u00e1rio, Av. Jos\u00e9 R. Sousa Fernandes, Lordem\u00e3o, Coimbra 3020-210, Portugal"},{"name":"Vetherapy\u2014Research and Development in Biotechnology, Coimbra 3020-210, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8114-0560","authenticated-orcid":false,"given":"Bart","family":"Spee","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht 3584, The Netherlands"}]}],"member":"54368","reference":[{"key":"2026032401350653000_2026.03.19.712113v1.1","doi-asserted-by":"publisher","DOI":"10.3390\/cells12081181"},{"key":"2026032401350653000_2026.03.19.712113v1.2","doi-asserted-by":"crossref","unstructured":"Penning LC , van den Boom R. Companion animal organoid technology to advance veterinary regenerative medicine. 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