Long‐Term Ex Vivo Maintenance of Functional Whole Prostate Glands from the Earthworm <i>Eudrilus eugeniae</i> and Its Applications in Regeneration, Drug Screening, and 3D Models

Prostate diseases are increasing globally, necessitating an appropriate model system for investigation. Ex vivo organ models help bridge the gap between organoids, assembloids, and in vivo models. This article represents a robust and reproducible approach for maintaining a functional ex vivo whole-organ prostate gland from the earthworm Eudrilus eugeniae, providing a unique platform for investigating organ-level regeneration, drug screening, and organoid-based research. Sexually mature E. eugeniae worms, which possess a pair of euprostate glands, are pre-processed, meticulously dissected in a sterile environment, and surface sterilized. The glands are treated sequentially in a series of buffers and antibiotic solutions and, without exposure to osmotic stress, are transferred to Leibovitz's L-15 medium containing 10% FBS. The ex vivo prostate glands are incubated at 23°C, simulating the organism's normal temperature. The viability and stress response of the gland are assessed through the rhythmic activity and frequency of prostatic fluids and cells discharged from the anterior duct. Microbial contamination is reduced through early detection and antibiotic treatment. Amputation of the posterior lobe facilitates regeneration via wound closure, contraction, and tissue remodeling, which are enhanced by the use of valproic acid. Released prostatic fluid facilitates noninvasive molecular diagnosis and monitoring of therapeutic response. The cell aggregates released into the medium from the posterior lobe form spheroids, which subsequently grow into organoids and mini-organ structures through self-assembly and cellular bridging. Overall, the developed ex vivo prostate model meets 3R principles for minimizing animal experimentation. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Pre-processing, dissection, and processing of prostate gland for ex vivo maintenance Support Protocol: Assessing ex vivo viability of prostate gland and handling contamination Basic Protocol 2: Functional ex vivo prostate gland regeneration, drug screening, and spheroid, organoid, and mini-organ formation.