Generation of Stickleback intestinal organoid v1

Intestinal organoids offer significant advantages over conventional 2D cultures, providing a physiologically relevant 3D architecture that enables more realistic disease modeling, host-microbe interactions, and host-pathogen studies. The threespine stickleback (Gasterosteus aculeatus) is an established teleost model widely used in evolutionary biology, adaptive immunity, and developmental genetics. However, robust long-term cell cultures from stickleback (whether fibroblast or epithelial in origin) have been challenging to establish, largely due to its cold water physiology and species-specific media requirements. Here, we present a first method for generating and maintaining intestinal organoids derived from juvenile stickleback. This protocol provides a novel experimental platform that is potentially adaptable to other teleost species. The resulting organoids are capable of expansion over multiple passages and are suitable for diverse downstream applications, including genetic, transcriptional, proteomic, and functional assays.