Method for modeling oviduct function and impact on embryonic development

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 Summary The protocol for generating mouse and human oviductal assembloids by combining epithelial and stromal cells for studying oviductal function in an in vitro setting.