{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T00:32:22Z","timestamp":1786408342212,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,7]]},"group-title":"Cell Biology","reference-count":28,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T00:00:00Z","timestamp":1786060800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"Eunice Kennedy Shriver National Institute of Child Health and Human Development","award":["R01HD112315"],"award-info":[{"award-number":["R01HD112315"]}]},{"name":"Eunice Kennedy Shriver National Institute of Child Health and Human Development","award":["R01HD097087"],"award-info":[{"award-number":["R01HD097087"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Direct experimental analysis of the mammalian oviduct is constrained by limited tissue access and the short lifespan of\n                  <jats:italic>ex vivo<\/jats:italic>\n                  preparations. Extracellular matrix-embedded three-dimensional epithelial organoids provide longer-term\n                  <jats:italic>in vitro<\/jats:italic>\n                  models. However, their inward-facing apical surface and the absence of supporting stromal cells limit physiological studies of the oviduct, including ciliary activity and maternal-embryonic interactions. Here, we provide a step-wise protocol detailing the generation of mouse and human oviductal assembloids in which epithelial cells form an outward-facing (apical-out) layer around a stromal core. Epithelial and stromal cells from adult mouse oviducts or human Fallopian tubes are isolated, expanded separately, and subsequently aggregated in a rotational culture system. The protocol also outlines morphological and immunostaining criteria for confirming cellular organization, whole-mount detection of external cilia, measurement of ciliary beat frequency, and co-culture of mouse assembloids with preimplantation embryos. Mouse and human assembloids retained epithelial and stromal identity and displayed cilia at the accessible outer surface. In a proof-of-concept experiment, embryos co-cultured with the assembloids developed to blastocysts at a rate similar to that of\n                  <jats:italic>in vivo<\/jats:italic>\n                  -derived blastocysts. This reductionist system provides a straightforward and tractable model to investigate oviduct physiology and embryo-maternal communication while allowing direct manipulation and observation of the epithelial interface.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"743297v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Summary<\/jats:title>\n                  <jats:p>\n                    The protocol for generating mouse and human oviductal assembloids by combining epithelial and stromal cells for studying oviductal function in an\n                    <jats:italic>in vitro<\/jats:italic>\n                    setting.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.08.06.743297","type":"posted-content","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T15:35:43Z","timestamp":1786116943000},"source":"Crossref","is-referenced-by-count":0,"title":["Method for modeling oviduct function and impact on embryonic development"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9323-3764","authenticated-orcid":false,"given":"Kalli K","family":"Stephens","sequence":"first","affiliation":[{"name":"Division of Animal Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, Missouri, USA 65211"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5071-8289","authenticated-orcid":false,"given":"Vakil","family":"Ahmad","sequence":"additional","affiliation":[{"name":"Department of Obstetrics, Gynecology, and Women\u2019s Health, University of Missouri, Columbia, Missouri, USA 65211"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria A.","family":"Silva","sequence":"additional","affiliation":[{"name":"Department of Obstetrics, Gynecology, and Women\u2019s Health, University of Missouri, Columbia, Missouri, USA 65211"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Makenna K","family":"Shifflett","sequence":"additional","affiliation":[{"name":"Department of Obstetrics, Gynecology, and Women\u2019s Health, University of Missouri, Columbia, Missouri, USA 65211"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0594-0571","authenticated-orcid":false,"given":"Jiude","family":"Mao","sequence":"additional","affiliation":[{"name":"Department of Obstetrics, Gynecology, and Women\u2019s Health, University of Missouri, Columbia, Missouri, USA 65211"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3855-1153","authenticated-orcid":false,"given":"Jason A.","family":"Rizo","sequence":"additional","affiliation":[{"name":"Division of Animal Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, Missouri, USA 65211"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8616-5003","authenticated-orcid":false,"given":"Mark I","family":"Hunter","sequence":"additional","affiliation":[{"name":"Department of Obstetrics, Gynecology, and Women\u2019s Health, University of Missouri, Columbia, Missouri, USA 65211"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1484-4998","authenticated-orcid":false,"given":"Andrew M","family":"Kelleher","sequence":"additional","affiliation":[{"name":"Department of Obstetrics, Gynecology, and Women\u2019s Health, University of Missouri, Columbia, Missouri, USA 65211"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5196-8471","authenticated-orcid":false,"given":"Wipawee","family":"Winuthayanon","sequence":"additional","affiliation":[{"name":"Division of Animal Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, Missouri, USA 65211"},{"name":"Department of Obstetrics, Gynecology, and Women\u2019s Health, University of Missouri, Columbia, Missouri, USA 65211"},{"name":"Department of Medical Pharmacology and Physiology, School of Medicine, University of Missouri, Columbia, Missouri, USA 65211"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026081017253127000_2026.08.06.743297v1.1","doi-asserted-by":"crossref","first-page":"383","DOI":"10.2527\/jas1965.242383x","article-title":"Collection of Secretions from the Oviduct and Uterus of the 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