Mouse Lacrimal Gland Ductal Organoids With Controlled Polarity: A Novel Ex Vivo Research Model

The aim of the present study was to develop a methodology for the culture and maintenance of ductal organoids derived from isolated lacrimal gland (LG) ducts, as well as to develop an approach for experimentally controlling epithelial polarity (apical-out [AO] versus apical-in [AI]).

Intra- and interlobular ducts were microdissected from the LG of FVB/N mice aged 12 to 24 weeks. Isolated ductal segments were embedded in Matrigel and expanded in defined media. AO polarity was achieved using baseline medium, whereas AI polarity was induced by a 3-day treatment with R-spondin, Wnt-3a, noggin, and Y-27632, followed by maintenance in baseline medium supplemented with forskolin. Epithelial polarity and ductal lineage identity were assessed by immunofluorescence staining for CFTR, AQP4, VPAC1/2, and NKCC1. Secretagogue-induced changes in organoid volume were quantified by video-microscopy.

Organoids formed within 7 to 10 days and were successfully passaged through passage 7 without detectable loss of morphological characteristics. CFTR exhibited apical localization, whereas AQP4 localized to the basolateral membrane. VPAC1/2 expression showed a mosaic pattern across passages. NKCC1 proteins were expressed predominantly on the basolateral surface of the organoids. In AO cultures, stimulation with secretagogues induced a significant reduction in the organoid projected area, consistent with net fluid secretion. In contrast, AI organoids exhibited stimulus-evoked alterations in luminal density without a corresponding change in the total projected area.

Duct-derived organoids retain key molecular features of native ductal epithelium, exhibit experimentally controllable epithelial polarity. This platform provides a versatile system for investigating ductal physiology and for the development of duct-targeted therapeutic strategies for dry eye disease.