Establishment of Mouse Meibomian Gland Organoids for In Vitro Modeling of Meibomian Gland Dysfunction

Purpose To establish a three-dimensional model of the mouse meibomian gland (MG) and to recapitulate meibomian gland dysfunction (MGD) for pathological investigations in MGD. Methods Primary mouse MG epithelial cells (MGECs) were embedded in Matrigel and expanded in serum-free medium, followed by the addition of extra growth factors to induce branching. Organoids were characterized by light microscopy, immunofluorescence, lipid staining, transmission electron microscopy, and RNA sequencing. The MGD organoid model was induced by interleukin-1β (IL-1β). Characteristics after orthotopic transplantation were assessed through hematoxylin and eosin and immunofluorescence staining. Results Mouse MGECs undergo spheroid formation, budding, and branching, forming organoids characterized by lipid- and debris-filled central lumens, surrounded by KRT6+ ductal cells, with Ki67+ progenitor cells present at early stages. The progenitor marker LRIG1, and the basal epithelial cell marker KRT5 were expressed throughout the organoids. Differentiation markers peroxisome proliferator-activated receptor gamma (PPARγ) and acyl‐CoA wax alcohol acyltransferase 2 (AWAT2) were detectable at days 7, 14, and 35. Transcriptomic analysis revealed upregulation of signaling pathways related to development in organoids. IL-1β exposure arrested organoid development, inhibited lipid accumulation, and induced hyperkeratinization of MG organoids, which was reversed by SB203580, an inhibitor of the p38 mitogen-activated protein kinase (MAPK) signaling pathway, or rosiglitazone, an agonist of the PPARγ signaling pathway. Transplanted organoids survived by 7 days with retained lipid droplets and ductal markers. Conclusions We developed mouse MG organoids that exhibited ductal–acinar architecture, sustained lipid production, and maintained stem/progenitor cell expression. Organoids modeled key features of MGD through exposure to IL-1β and can respond to targeted therapy. The system offered a promising platform for MGD research.