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bioRxiv · 10.64898/2026.08.06.743297

Method for modeling oviduct function and impact on embryonic development

Stephens, K. K.·Ahmad, V.·Silva, M. A.·Shifflett, M. K.·Mao, J.·Rizo, J. A.·Hunter, M. I.·Kelleher, A. M.·Winuthayanon, W.
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Abstract

Direct experimental analysis of the mammalian oviduct is constrained by limited tissue access and the short lifespan of ex vivo preparations. Extracellular matrix-embedded three-dimensional epithelial organoids provide longer-term in vitro 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 in vivo-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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/743297v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1acb462org.highwire.dtl.DTLVardef@17695f8org.highwire.dtl.DTLVardef@6fd6c3org.highwire.dtl.DTLVardef@7cc470_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryThe protocol for generating mouse and human oviductal assembloids by combining epithelial and stromal cells for studying oviductal function in an in vitro setting.

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Stephens, K. K., Ahmad, V., Silva, M. A., Shifflett, M. K., Mao, J., Rizo, J. A., Hunter, M. I., Kelleher, A. M., Winuthayanon, W.. 2026-08-07. Method for modeling oviduct function and impact on embryonic development. https://doi.org/10.64898/2026.08.06.743297

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