Protocol for scalable generation of uniform 3D porcine oviduct epithelial cell spheroids

While previous in vitro porcine (Sus scrofa) oviduct spheroid models often rely on manual selection of the formed aggregates, here, we establish a scalable and reproducible system for generating oviduct spheroids. We describe steps for isolating primary secretory and ciliated oviduct epithelial cells and their assembling them into spheroids by forced aggregation using the AggreWell platform. This enables the mass production of uniformly sized spheroids ready for use in a range of downstream applications, such as sperm-oviduct co-incubation experiments. For complete details on the use and execution of this protocol, please refer to Molina et al.1 Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/731731v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@20f94aorg.highwire.dtl.DTLVardef@ce80eeorg.highwire.dtl.DTLVardef@172c631org.highwire.dtl.DTLVardef@31f420_HPS_FORMAT_FIGEXP M_FIG C_FIG Before you beginThis protocol describes the isolation of primary porcine oviduct epithelial cells and their use in generating a three-dimensional (3D) spheroid model in vitro. The protocol is specifically designed to enrich and preserve ciliated oviduct epithelial cells, which are critical for physiologically relevant sperm-oviduct interaction studies yet are frequently underrepresented in conventional in vitro oviduct models. To minimize the de-differentiation of ciliated cells during culture, a specialized Incubation Medium is employed to maintain epithelial cell viability and ciliary function. The relatively brief spheroid formation period (48 h) further supports preservation of ciliary structure and activity prior to downstream functional applications. This protocol consists of four major steps. Steps 1 and 2 cover oviductal epithelial cell isolation through mechanical and enzymatic digestion, step 3 describes the spheroid formation and culture conditions, and step 4 applies the spheroid model to sperm co-incubations or immunofluorescent staining. Although optimized for the porcine isthmus, this protocol may also be suitable for the cells from the ampulla and adaptable to other large animal species.