AbstractBackground and ObjectivesFew preclinical models of pseudomyxoma peritonei (PMP) have been developed, probably due to the tumor's low incidence and its peculiar characteristics of slow growth. Therefore, there is a need to develop more refined PMP models that better reflect its characteristics. The aim of the study is to develop a culture strategy to generate organoid models derived from PMP patient samples.MethodsWe followed a strategy based on combinatorial culture conditions that include the different factors essential for PMP growth and that mimic the microenvironment present in the patients.ResultsWe cultured PMP samples in the presence of the various factors produced by the niche environment of PMP. We obtained 12 PMP organoid models, each of which grows under specific culture conditions. PMP‐derived organoids show long‐term expansion capacity and reproduce the genetic landscape and histological phenotype of the tumor of origin.ConclusionThe organoids we developed faithfully reproduce the key features of PMP disease and will allow us to understand the biology of PMP. With them, we will be able to identify key regulatory networks that support PMP progression, providing a platform for multilevel preclinical testing, identify novel diagnostic biomarkers, and generate novel targets for patient treatments.