Epithelial ion and fluid secretion determine the physiological functions of a broad range of organs, such as the lung, liver, or pancreas. The molecular mechanism of pancreatic ion secretion is challenging to investigate due to the limited access to functional human ductal epithelia. Patient-derived organoids may overcome these limitations, however direct accessibility of the apical membrane is not solved. In addition, due to the vectorial transport of ions and fluid the intraluminal pressure in the organoids is elevated, which may hinder the study of physiological processes. To overcome these, we developed an advanced culturing method for human pancreatic organoids based on the removal of the extracellular matrix that induced an apical-to-basal polarity switch also leading to reversed localization of proteins with polarized expression. The cells in the apical-out organoids had a cuboidal shape, whereas their resting intracellular Ca2+concentration was more consistent compared to the cells in the apical-in organoids. Using this advanced model, we demonstrated the expression and function of two novel ion channels, the Ca2+activated Cl−channel Anoctamin 1 (ANO1) and the epithelial Na+channel (ENaC), which were not considered in ductal cells yet. Finally, we showed that the available functional assays, such as forskolin-induced swelling, or intracellular Cl−measurement have improved dynamic range when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1007/s00018-023-04836-2.