The mammary gland is a dynamic organ whose parenchyma undergoes major development during puberty and extensive remodeling with each estrous cycle. These processes can be modelled and investigated in vitro via 3D cell culture techniques that employ specialized extracellular matrices and appropriate growth factors. The resulting mammary organoid cultures faithfully represent the mammary gland with respect to cellular heterogeneity, cell-cell contacts, overall architecture as well as response to growth factor stimuli and are amendable to a variety of molecular methods as well as microscopy techniques. Among the imaging techniques, light sheet microscopy (single plane illumination microscopy; SPIM) represents a useful method for longitudinal monitoring of morphological changes and cell behavior during the establishment of mammary gland ductal systems. In contrast to other fluorescence microscopy techniques such as widefield- and confocal-microscopy, SPIM exerts minimal phototoxicity while allowing fast acquisition of different fluorophores within organoids arranged in a 3D matrix under optimized environmental conditions. Here, we provide a detailed protocol for organoid acquisition and culture and describe two sample mounting variants for use with multiview and inverted light sheet microscopes.

• Keywords
• 3D cell culture, Epithelium, Light sheet microscopy, Mammary gland, Time-lapse,
• Publication type
• Journal Article MeSH

The collection on Methods and Models in Mammary Gland Biology and Breast Cancer Research showcases recent advances in tools and models that enhance our understanding of mammary gland development and breast cancer. This collection includes sixteen articles, collectively addressing approaches to investigate key aspects of mammary gland biology and tumorigenesis, including hormonal signaling, tissue architecture, tumor microenvironment, and species-specific mammary development. The issue highlights innovations such as optimized progesterone receptor reporters, improved menopause models, and 3D-printed mammary epithelial structures. It also features advancements in organoid-based studies, in situ labeling of epithelial proliferation in large animals, preclinical models for breast cancer prevention, and high-resolution imaging techniques. Methodologies for studying macrophage-cancer cell interactions and lysosomal function are provided as step-by-step protocols. Additionally, review articles provide insights into diverse mammalian organoid systems, rat mammary tumor models, and strategies for modeling breast cancer metastasis. Together, these contributions advance mammary gland research by refining experimental approaches, expanding model diversity, and fostering translational applications in breast cancer.

• MeSH
• Rats MeSH
• Humans MeSH
• Mammary Glands, Human * pathology   metabolism   MeSH
• Mammary Glands, Animal * pathology   metabolism   MeSH
• Disease Models, Animal MeSH
• Tumor Microenvironment MeSH
• Breast Neoplasms * pathology   metabolism   MeSH
• Organoids MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Introductory Journal Article MeSH
• Editorial MeSH