Establishment and characterization study of ovine mammary organoids

The study of ovine lactation and mastitis is limited by the lack of physiologically relevant in vitro models. Compared to mammary epithelial cells, mammary gland organoids (MaOs) offer a better model for studying lactation, as they more accurately replicate the tissue structure and interactions of cells in vivo. Here, we optimize a protocol for the isolation, culture, characterization, and transfection of ovine MaOs derived from ewe mammary tissue. Results: We compared different enzymatic digestion methods and found that type IV collagenase digestion for 60 min yielded the highest number of MaOs compared to a mixed enzyme solution of type II collagenase and hyaluronidase. Culture medium optimization revealed that medium B supported superior growth, with increased budding structures, a higher number of MaOs over time, and a greater proportion of EdU-positive proliferating cells compared to media A and C. Cell-type characterization confirmed the presence of both luminal and myoepithelial cells, as shown by RT-PCR analysis of CK14, CK18, and CK7 expression, and further validated by immunofluorescence staining for CK14 and CK18. MaOs in medium B exhibited significantly higher mRNA expression levels of milk lipid metabolism-related genes (XDH, FABP3, SREBP1), lactose metabolism-related genes (GLUT1, GLUT4), and milk protein synthesis genes (EIF4E, CSN2) at multiple time points. In all media, XDH, FABP3, SREBP1, GLUT1, GLUT4, EIF4E, and CSN2 mRNA expression peaked at Day 7 before declining. Additionally, at Day 7, supernatant analysis confirmed that triglyceride, lactose, and CSN2 concentrations were significantly elevated in medium B compared to media A and C. Finally, we demonstrated that the ovine MaOs could be transfected using electroporation with 27.3% becoming GFP-positive. Conclusions: This study establishes a protocol for the isolation, culture, characterization, and genetic manipulation of ovine MaOs. These organoids serve as a physiologically relevant model for studying the regulatory mechanisms of lactation and mastitis in sheep, providing a tool for future research in veterinary and agricultural sciences.