{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T11:16:16Z","timestamp":1770894976039,"version":"3.50.1"},"reference-count":45,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2019,10,25]],"date-time":"2019-10-25T00:00:00Z","timestamp":1571961600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002873","name":"Chulalongkorn University","doi-asserted-by":"publisher","award":["DNS_62_003_32_001_1"],"award-info":[{"award-number":["DNS_62_003_32_001_1"]}],"id":[{"id":"10.13039\/501100002873","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002873","name":"Chulalongkorn University","doi-asserted-by":"publisher","award":["STAR 2019"],"award-info":[{"award-number":["STAR 2019"]}],"id":[{"id":"10.13039\/501100002873","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001349","name":"National Medical Research Council","doi-asserted-by":"publisher","award":["NMRC\/CNIG\/1131\/2015"],"award-info":[{"award-number":["NMRC\/CNIG\/1131\/2015"]}],"id":[{"id":"10.13039\/501100001349","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Faculty of Dentistry, Chulalongkorn University","award":["Special Funding 2018"],"award-info":[{"award-number":["Special Funding 2018"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Research efforts have been made to develop human salivary gland (SG) secretory epithelia for transplantation in patients with SG hypofunction and dry mouth (xerostomia). However, the limited availability of human biopsies hinders the generation of sufficient cell numbers for epithelia formation and regeneration. Porcine SG have several similarities to their human counterparts, hence could replace human cells in SG modelling studies in vitro. Our study aims to establish porcine SG explant outgrowth models to generate functional secretory epithelia for regeneration purposes to rescue hyposalivation. Cells were isolated and expanded from porcine submandibular and parotid gland explants. Flow cytometry, immunocytochemistry, and gene arrays were performed to assess proliferation, standard mesenchymal stem cell, and putative SG epithelial stem\/progenitor cell markers. Epithelial differentiation was induced and different SG-specific markers investigated. Functional assays upon neurostimulation determined \u03b1-amylase activity, trans-epithelial electrical resistance, and calcium influx. Primary cells exhibited SG epithelial progenitors and proliferation markers. After differentiation, SG markers were abundantly expressed resembling epithelial lineages (E-cadherin, Krt5, Krt14), and myoepithelial (\u03b1-smooth muscle actin) and neuronal (\u03b23-tubulin, Chrm3) compartments. Differentiated cells from submandibular gland explant models displayed significantly greater proliferation, number of epithelial progenitors, amylase activity, and epithelial barrier function when compared to parotid gland models. Intracellular calcium was mobilized upon cholinergic and adrenergic neurostimulation. In summary, this study highlights new strategies to develop secretory epithelia from porcine SG explants, suitable for future proof-of-concept SG regeneration studies, as well as for testing novel muscarinic agonists and other biomolecules for dry mouth.<\/jats:p>","DOI":"10.3390\/biom9110657","type":"journal-article","created":{"date-parts":[[2019,10,25]],"date-time":"2019-10-25T11:05:18Z","timestamp":1572001518000},"page":"657","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Strategies for Developing Functional Secretory Epithelia from Porcine Salivary Gland Explant Outgrowth Culture Models"],"prefix":"10.3390","volume":"9","author":[{"given":"Ganokon","family":"Urkasemsin","sequence":"first","affiliation":[{"name":"Department of Preclinical and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Phoebe","family":"Castillo","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry, National University of Singapore, Singapore 119085, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sasitorn","family":"Rungarunlert","sequence":"additional","affiliation":[{"name":"Department of Preclinical and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nuttha","family":"Klincumhom","sequence":"additional","affiliation":[{"name":"Exocrine Gland Biology and Regeneration Research Group, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand"},{"name":"Center of Excellence in Regenerative Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4230-4593","authenticated-orcid":false,"given":"Joao N.","family":"Ferreira","sequence":"additional","affiliation":[{"name":"Exocrine Gland Biology and Regeneration Research Group, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand"},{"name":"Center of Excellence in Regenerative Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,10,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2142","DOI":"10.1111\/jgs.13652","article-title":"Meta-Analysis of Salivary Flow Rates in Young and Older Adults","volume":"63","author":"Affoo","year":"2015","journal-title":"J. Am. Geriatr. Soc."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"298","DOI":"10.14219\/jada.archive.1989.0211","article-title":"The role of saliva in maintaining oral homeostasis","volume":"119","author":"Mandel","year":"1989","journal-title":"J. Am. Dent. Assoc."},{"key":"ref_3","first-page":"S57e1","article-title":"Salivary dysfunction associated with systemic diseases: Systematic review and clinical management recommendations","volume":"103","author":"Sollecito","year":"2007","journal-title":"Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1061","DOI":"10.1007\/s00520-010-0837-6","article-title":"A systematic review of salivary gland hypofunction and xerostomia induced by cancer therapies: Management strategies and economic impact","volume":"18","author":"Jensen","year":"2010","journal-title":"Support. Care Cancer"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"894","DOI":"10.1177\/0022034509343143","article-title":"Sensitivity of salivary glands to radiation: From animal models to therapies","volume":"88","author":"Grundmann","year":"2009","journal-title":"J. Dent. Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"983","DOI":"10.1016\/j.ijrobp.2010.06.052","article-title":"Clinical management of salivary gland hypofunction and xerostomia in head-and-neck cancer patients: Successes and barriers","volume":"78","author":"Vissink","year":"2010","journal-title":"Int. J. Radiat. Oncol. Biol. Phys."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"958","DOI":"10.1097\/01.MLG.0000163340.90211.87","article-title":"Chemoradiation-induced cell loss in human submandibular glands","volume":"115","author":"Sullivan","year":"2005","journal-title":"Laryngoscope"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1016\/S0959-8049(05)80076-0","article-title":"Parotid gland function during and following radiotherapy of malignancies in the head and neck. A consecutive study of salivary flow and patient discomfort","volume":"28","author":"Franzen","year":"1992","journal-title":"Eur. J. Cancer"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1067\/mpr.2002.128176","article-title":"The impact of saliva on patient care: A literature review","volume":"88","author":"Marek","year":"2002","journal-title":"J. Prosthet. Dent."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1096","DOI":"10.1002\/j.0022-0337.2001.65.10.tb03455.x","article-title":"Salivary enhancement: Current status and future therapies","volume":"65","author":"Atkinson","year":"2001","journal-title":"J. Dent. Educ."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"110","DOI":"10.5966\/sctm.2016-0083","article-title":"Primary Salivary Human Stem\/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture","volume":"6","author":"Srinivasan","year":"2017","journal-title":"Stem Cells Transl. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1016\/j.actbio.2017.08.009","article-title":"Elastin-PLGA hybrid electrospun nanofiber scaffolds for salivary epithelial cell self-organization and polarization","volume":"62","author":"Foraida","year":"2017","journal-title":"Acta Biomater."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"640","DOI":"10.1002\/stem.2278","article-title":"Human Salivary Gland Stem Cells Functionally Restore Radiation Damaged Salivary Glands","volume":"34","author":"Pringle","year":"2016","journal-title":"Stem Cells"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.biomaterials.2018.06.011","article-title":"Engineering innervated secretory epithelial organoids by magnetic three-dimensional bioprinting for stimulating epithelial growth in salivary glands","volume":"180","author":"Adine","year":"2018","journal-title":"Biomaterials"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1111\/odi.12840","article-title":"Comparing human and mouse salivary glands: A practice guide for salivary researchers","volume":"25","author":"Maruyama","year":"2019","journal-title":"Oral Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1016\/j.radonc.2015.06.022","article-title":"Similar ex vivo expansion and post-irradiation regenerative potential of juvenile and aged salivary gland stem cells","volume":"116","author":"Maimets","year":"2015","journal-title":"Radiother. Oncol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1002\/term.1952","article-title":"Three-dimensional cultures of mouse submandibular and parotid glands: A comparative study","volume":"11","author":"Leigh","year":"2017","journal-title":"J. Tissue Eng. Regen. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"530","DOI":"10.1111\/j.1601-0825.2006.01337.x","article-title":"The miniature pig: A useful large animal model for dental and orofacial research","volume":"13","author":"Wang","year":"2007","journal-title":"Oral Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1038\/sj.dmfr.4600336","article-title":"Sialographic characterization of the normal parotid gland of the miniature pig","volume":"27","author":"Wang","year":"1998","journal-title":"Dentomaxillofac. Radiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1016\/S1079-2104(03)00369-X","article-title":"Structural and functional injury in minipig salivary glands following fractionated exposure to 70 Gy of ionizing radiation: An animal model for human radiation-induced salivary gland injury","volume":"96","author":"Radfar","year":"2003","journal-title":"Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1002\/jgm.1081","article-title":"Sorting of transgenic secretory proteins in miniature pig parotid glands following adenoviral-mediated gene transfer","volume":"9","author":"Yan","year":"2007","journal-title":"J. Gene Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1111\/joa.12833","article-title":"Expression patterns of tight junction proteins in porcine major salivary glands: A comparison study with human and murine glands","volume":"233","author":"Zhang","year":"2018","journal-title":"J. Anat."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1002\/stem.2455","article-title":"Concise Review: Salivary Gland Regeneration: Therapeutic Approaches from Stem Cells to Tissue Organoids","volume":"35","author":"Lombaert","year":"2017","journal-title":"Stem Cells"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1002\/term.2809","article-title":"A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids","volume":"13","author":"Ferreira","year":"2019","journal-title":"J. Tissue Eng. Regen. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1016\/j.ymthe.2004.11.007","article-title":"Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands","volume":"11","author":"Shan","year":"2005","journal-title":"Mol. Ther."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1016\/j.omtm.2018.02.008","article-title":"Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction","volume":"9","author":"Ferreira","year":"2018","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1408","DOI":"10.1002\/stem.1681","article-title":"Concise review: The surface markers and identity of human mesenchymal stem cells","volume":"32","author":"Lv","year":"2014","journal-title":"Stem Cells"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1111\/odi.12812","article-title":"CD29 is highly expressed on epithelial, myoepithelial, and mesenchymal stromal cells of human salivary glands","volume":"24","author":"Togarrati","year":"2018","journal-title":"Oral Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"957","DOI":"10.1016\/j.stemcr.2014.09.015","article-title":"Purification and ex vivo expansion of fully functional salivary gland stem cells","volume":"3","author":"Nanduri","year":"2014","journal-title":"Stem Cell Rep."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1111\/j.1601-0825.2011.01858.x","article-title":"Cell surface markers CD44 and CD166 localized specific populations of salivary acinar cells","volume":"18","author":"Maria","year":"2012","journal-title":"Oral Dis."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"36303","DOI":"10.1038\/srep36303","article-title":"Single Cell Clones Purified from Human Parotid Glands Display Features of Multipotent Epitheliomesenchymal Stem Cells","volume":"6","author":"Yi","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"10106","DOI":"10.1038\/srep10106","article-title":"Characterization of a Self-renewing and Multi-potent Cell Population Isolated from Human Minor Salivary Glands","volume":"5","author":"Lu","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"776","DOI":"10.1177\/0022034516634664","article-title":"Identification of Stem Cells in the Secretory Complex of Salivary Glands","volume":"95","author":"Kwak","year":"2016","journal-title":"J. Dent. Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1502","DOI":"10.1177\/0022034515599770","article-title":"Salivary Glands: Stem Cells, Self-duplication, or Both?","volume":"94","author":"Aure","year":"2015","journal-title":"J. Dent. Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1007\/978-1-4939-2519-3_2","article-title":"A protocol for studying embryonic mammary progenitor cells during mouse mammary primordial development in explant culture","volume":"1293","author":"Kogata","year":"2015","journal-title":"Methods Mol. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1111\/exd.12712","article-title":"Ex vivo human skin and SZ95 sebocytes exhibit a homoeostatic interaction in a novel coculture contact model","volume":"24","author":"Nikolakis","year":"2015","journal-title":"Exp. Dermatol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"304","DOI":"10.1177\/0022034514559251","article-title":"Establishment of functional acinar-like cultures from human salivary glands","volume":"94","author":"Jang","year":"2015","journal-title":"J. Dent. Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1510","DOI":"10.1016\/j.ijrobp.2005.04.029","article-title":"Structural and functional characteristics of irradiation damage to parotid glands in the miniature pig","volume":"62","author":"Li","year":"2005","journal-title":"Int. J. Radiat. Oncol. Biol. Phys."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"506","DOI":"10.1002\/jgm.1319","article-title":"Long-term transduction of miniature pig parotid glands using serotype 2 adeno-associated viral vectors","volume":"11","author":"Hai","year":"2009","journal-title":"J. Gene Med."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4321","DOI":"10.7150\/thno.26509","article-title":"Intragland Shh gene delivery mitigated irradiation-induced hyposalivation in a miniature pig model","volume":"8","author":"Hu","year":"2018","journal-title":"Theranostics"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1089\/clo.2006.0022","article-title":"Isolation of tissue progenitor cells from duct-ligated salivary glands of swine","volume":"9","author":"Matsumoto","year":"2007","journal-title":"Cloning Stem Cells"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1038\/sj.bdj.4807284","article-title":"Saliva and dental health. Clinical implications of saliva: Report of a consensus meeting","volume":"169","author":"Edgar","year":"1990","journal-title":"Br. Dent. J."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1","DOI":"10.15436\/2471-0598.16.013","article-title":"Neurons Self-Organize Around Salivary Epithelial Cells in Novel Co-Culture Model","volume":"2","author":"Sommakia","year":"2016","journal-title":"J. Stem Cell Regen. Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1016\/j.stemcr.2015.11.009","article-title":"Long-Term In Vitro Expansion of Salivary Gland Stem Cells Driven by Wnt Signals","volume":"6","author":"Maimets","year":"2016","journal-title":"Stem Cell Rep."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1135","DOI":"10.1089\/ten.tec.2009.0438","article-title":"Rat parotid gland cell differentiation in three-dimensional culture","volume":"16","author":"Baker","year":"2010","journal-title":"Tissue Eng. Part C Methods"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/9\/11\/657\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:29:30Z","timestamp":1760189370000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/9\/11\/657"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,10,25]]},"references-count":45,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2019,11]]}},"alternative-id":["biom9110657"],"URL":"https:\/\/doi.org\/10.3390\/biom9110657","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,10,25]]}}}