{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T05:58:41Z","timestamp":1792648721961,"version":"4.5.0"},"reference-count":41,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2021,7,16]],"date-time":"2021-07-16T00:00:00Z","timestamp":1626393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100014579","name":"National Institute of Animal Science","doi-asserted-by":"publisher","award":["PJ01422201"],"award-info":[{"award-number":["PJ01422201"]}],"id":[{"id":"10.13039\/501100014579","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Intestinal organoids offer great promise for disease-modelling-based host\u2013pathogen interactions and nutritional research for feed efficiency measurement in livestock and regenerative medicine for therapeutic purposes. However, very limited studies are available on the functional characterisation and three-dimensional (3D) expansion of adult stem cells in livestock species compared to other species. Intestinal crypts derived from intestinal organoids under a 3D culture system from the small intestine in adult bovine were successfully established and characterised for functionality testing, including the cellular potentials and genetic properties based on immunohistochemistry, immunocytochemistry, epithelial barrier permeability assay, QuantSeq 3\u2032 mRNA-Seq. data and quantitative reverse transcription-polymerase chain reaction. Intestinal organoids were long-term cultivated over several passages of culture without loss of the recapitulating capacity of crypts, and they had the specific expression of several specific markers involved in intestinal stem cells, intestinal epithelium, and nutrient absorption. In addition, they showed the key functionality with regard to a high permeability for compounds of up to FITC-dextran 4 kDa, while FITC-dextran 40 kDa failed to enter the organoid lumen and revealed that the genetic properties of bovine intestinal organoids were highly similar to those of in vivo. Collectively, these results provide a reliable method for efficient isolation of intestinal crypts from the small intestine and robust 3D expansion of intestinal organoids in adult bovine and demonstrate the in vitro 3D organoids mimics the in vivo tissue topology and functionality. Finally, intestinal organoids are potential alternatives to in vivo systems and will be facilitated as the practical model to replace animal experiments for various purposes in the fields of animal biotechnology.<\/jats:p>","DOI":"10.3390\/ani11072115","type":"journal-article","created":{"date-parts":[[2021,7,16]],"date-time":"2021-07-16T10:52:58Z","timestamp":1626432778000},"page":"2115","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Robust Three-Dimensional (3D) Expansion of Bovine Intestinal Organoids: An In Vitro Model as a Potential Alternative to an In Vivo System"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0537-6205","authenticated-orcid":false,"given":"Bo-Ram","family":"Lee","sequence":"first","affiliation":[{"name":"Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4162-4410","authenticated-orcid":false,"given":"Hyeon","family":"Yang","sequence":"additional","affiliation":[{"name":"Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sang-In","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Animal Biotechnology, Kyungpook National University, Sangju-si 37224, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0661-2203","authenticated-orcid":false,"given":"Inamul","family":"Haq","sequence":"additional","affiliation":[{"name":"Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sun-A","family":"Ock","sequence":"additional","affiliation":[{"name":"Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hayeon","family":"Wi","sequence":"additional","affiliation":[{"name":"Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hwi-Cheul","family":"Lee","sequence":"additional","affiliation":[{"name":"Planning and Coordination Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2447-0392","authenticated-orcid":false,"given":"Poongyeon","family":"Lee","sequence":"additional","affiliation":[{"name":"Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8542-9193","authenticated-orcid":false,"given":"Jae-Gyu","family":"Yoo","sequence":"additional","affiliation":[{"name":"Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"590","DOI":"10.1016\/j.devcel.2016.08.014","article-title":"Organoids: Modeling Development and the Stem Cell Niche in a Dish","volume":"38","author":"Kretzschmar","year":"2016","journal-title":"Dev. Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/j.biomaterials.2018.12.006","article-title":"Intestinal organoids: A new paradigm for engineering intestinal epithelium in vitro","volume":"194","author":"Rahmani","year":"2019","journal-title":"Biomaterials"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1097\/MPG.0000000000001411","article-title":"Intestinal Organoids: New Frontiers in the Study of Intestinal Disease and Physiology","volume":"64","author":"Wallach","year":"2017","journal-title":"J. Pediatr. Gastroenterol. Nutr."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"375","DOI":"10.3389\/fcell.2020.00375","article-title":"Congruence of Transcription Programs in Adult Stem Cell-Derived Jejunum Organoids and Original Tissue During Long-Term Culture","volume":"8","author":"Madsen","year":"2020","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2509","DOI":"10.1016\/j.celrep.2019.01.108","article-title":"Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions","volume":"26","author":"Co","year":"2019","journal-title":"Cell Rep."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"skaa011","DOI":"10.1093\/jas\/skaa011","article-title":"Evaluation of swine enteroids as in vitro models for Lawsonia intracellularis infection","volume":"98","author":"Resende","year":"2020","journal-title":"J. Anim. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1186\/s13567-021-00909-x","article-title":"Intestinal organoids in farm animals","volume":"52","author":"Beaumont","year":"2021","journal-title":"Vet. Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1186\/s13567-021-00904-2","article-title":"Organoids: A promising new in vitro platform in livestock and veterinary research","volume":"52","author":"Kar","year":"2021","journal-title":"Vet. Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1186\/s13567-018-0547-5","article-title":"Development of in vitro enteroids derived from bovine small intestinal crypts","volume":"49","author":"Hamilton","year":"2018","journal-title":"Vet. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e01006-20","DOI":"10.1128\/JVI.01006-20","article-title":"Next-Generation Porcine Intestinal Organoids: An Apical-Out Organoid Model for Swine Enteric Virus Infection and Immune Response Investigations","volume":"94","author":"Li","year":"2020","journal-title":"J. Virol."},{"key":"ref_11","first-page":"255","article-title":"In vitro demonstration of intestinal absorption mechanisms of different sugars using 3D organotypic tissues in a fluidic device","volume":"37","author":"Marrella","year":"2020","journal-title":"ALTEX"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2806","DOI":"10.1111\/cas.14118","article-title":"Establishment of a novel experimental model for muscle-invasive bladder cancer using a dog bladder cancer organoid culture","volume":"110","author":"Elbadawy","year":"2019","journal-title":"Cancer Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"119823","DOI":"10.1016\/j.biomaterials.2020.119823","article-title":"Efficacy of primary liver organoid culture from different stages of non-alcoholic steatohepatitis (NASH) mouse model","volume":"237","author":"Elbadawy","year":"2020","journal-title":"Biomaterials"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"9393","DOI":"10.1038\/s41598-020-66229-w","article-title":"Establishment of 2.5D organoid culture model using 3D bladder cancer organoid culture","volume":"10","author":"Abugomaa","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2329","DOI":"10.1038\/nprot.2014.158","article-title":"Generation of cerebral organoids from human pluripotent stem cells","volume":"9","author":"Lancaster","year":"2014","journal-title":"Nat. Protoc."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1038\/nature07935","article-title":"Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche","volume":"459","author":"Sato","year":"2009","journal-title":"Nature"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1007\/s13770-020-00278-0","article-title":"Evaluation of Intestinal Epithelial Barrier Function in Inflammatory Bowel Diseases Using Murine Intestinal Organoids","volume":"17","author":"Rallabandi","year":"2020","journal-title":"Tissue Eng. Regen. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1007\/s00441-018-2924-9","article-title":"Developing a 3D intestinal epithelium model for livestock species","volume":"375","author":"Derricott","year":"2019","journal-title":"Cell Tissue Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1762","DOI":"10.1053\/j.gastro.2011.07.050","article-title":"Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett\u2019s epithelium","volume":"141","author":"Sato","year":"2011","journal-title":"Gastroenterology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1073\/pnas.1118857109","article-title":"The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations","volume":"109","author":"Yan","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3710836","DOI":"10.1155\/2016\/3710836","article-title":"Three-Dimensional Gastrointestinal Organoid Culture in Combination with Nerves or Fibroblasts: A Method to Characterize the Gastrointestinal Stem Cell Niche","volume":"2016","author":"Pastula","year":"2016","journal-title":"Stem Cells Int."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"173","DOI":"10.12750\/JARB.34.3.173","article-title":"Effects of short-term acute heat stress on physiological responses and heat shock proteins of Hanwoo steer (Korean cattle)","volume":"34","author":"Baek","year":"2019","journal-title":"J. Anim. Reprod. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1007\/s00418-009-0648-3","article-title":"Characterization of newly established bovine intestinal epithelial cell line","volume":"133","author":"Miyazawa","year":"2010","journal-title":"Histochem. Cell Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1007\/s11626-016-0082-5","article-title":"Establishment of primary bovine intestinal epithelial cell culture and clone method","volume":"53","author":"Zhan","year":"2017","journal-title":"In Vitro Cell. Dev. Biol. Anim."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1001","DOI":"10.1096\/fj.201901294R","article-title":"A novel F-box domain containing cyclin F like gene is required for maintaining the genome stability and survival of chicken primordial germ cells","volume":"34","author":"Lee","year":"2020","journal-title":"FASEB J."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Lee, B.R., Kim, H., Park, T.S., Moon, S., Cho, S., Park, T., Lim, J.M., and Han, J.Y. (2007). A set of stage-specific gene transcripts identified in EK stage X and HH stage 3 chick embryos. BMC Dev. Biol., 7.","DOI":"10.1186\/1471-213X-7-60"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/nmeth.1923","article-title":"Fast gapped-read alignment with Bowtie 2","volume":"9","author":"Langmead","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1093\/bioinformatics\/btq033","article-title":"BEDTools: A flexible suite of utilities for comparing genomic features","volume":"26","author":"Quinlan","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"R80","DOI":"10.1186\/gb-2004-5-10-r80","article-title":"Bioconductor: Open software development for computational biology and bioinformatics","volume":"5","author":"Gentleman","year":"2004","journal-title":"Genome Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"104928","DOI":"10.1016\/j.tiv.2020.104928","article-title":"Human ileal organoid model recapitulates clinical incidence of diarrhea associated with small molecule drugs","volume":"68","author":"Belair","year":"2020","journal-title":"Toxicol. Vitr."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"307","DOI":"10.2144\/0000113851","article-title":"Prostaglandin E2 supports growth of chicken embryo intestinal organoids in Matrigel matrix","volume":"52","author":"Pierzchalska","year":"2012","journal-title":"Biotechniques"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2926","DOI":"10.1128\/IAI.00161-15","article-title":"Interaction of Salmonella enterica Serovar Typhimurium with Intestinal Organoids Derived from Human Induced Pluripotent Stem Cells","volume":"83","author":"Forbester","year":"2015","journal-title":"Infect. Immun."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1128\/JVI.01930-15","article-title":"Human Intestinal Enteroids: A New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology","volume":"90","author":"Saxena","year":"2016","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1038\/nri3608","article-title":"Intestinal epithelial cells: Regulators of barrier function and immune homeostasis","volume":"14","author":"Peterson","year":"2014","journal-title":"Nat. Rev. Immunol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Pearce, S.C., Al-Jawadi, A., Kishida, K., Yu, S., Hu, M., Fritzky, L.F., Edelblum, K.L., Gao, N., and Ferraris, R.P. (2018). Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types. BMC Biol., 16.","DOI":"10.1186\/s12915-018-0481-z"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"734","DOI":"10.1016\/j.stem.2013.09.015","article-title":"Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury","volume":"13","author":"Fordham","year":"2013","journal-title":"Cell Stem Cell"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"787","DOI":"10.1016\/j.stem.2018.11.016","article-title":"Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition","volume":"23","author":"Fujii","year":"2018","journal-title":"Cell Stem Cell"},{"key":"ref_39","first-page":"56960","article-title":"Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids","volume":"130","author":"Hill","year":"2017","journal-title":"J. Vis. Exp."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2819154","DOI":"10.1155\/2018\/2819154","article-title":"Regulation of Intestinal Epithelial Cells Properties and Functions by Amino Acids","volume":"2018","author":"Kong","year":"2018","journal-title":"Biomed. Res. Int."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"16831","DOI":"10.1038\/srep16831","article-title":"Intestinal organoids for assessing nutrient transport, sensing and incretin secretion","volume":"5","author":"Zietek","year":"2015","journal-title":"Sci. Rep."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/11\/7\/2115\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:31:25Z","timestamp":1760164285000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/11\/7\/2115"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,16]]},"references-count":41,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2021,7]]}},"alternative-id":["ani11072115"],"URL":"https:\/\/doi.org\/10.3390\/ani11072115","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,16]]}}}