A Defined Hydrogel-Based Method For Generating Three-Dimensional Human Breast Organoids That Recapitulate Mammary Morphogenesis.

The development of physiologically relevant human model systems that recapitulate tissue architecture and cell-state dynamics remains a major challenge in studying breast development and early events in carcinogenesis. Conventional two-dimensional cultures and many three-dimensional systems fail to capture the structural organization and microenvironmental cues that define the human mammary gland. Here, we describe a reproducible method for generating three-dimensional human breast organoids from primary epithelial cells embedded within a defined hydrogel matrix composed of type I collagen, laminin, fibronectin, and hyaluronic acid. This system supports the progression of single cells through key stages of mammary morphogenesis, including progenitor expansion, epithelial patterning, and the formation of terminal ductal lobular unit-like structures, as well as the emergence of a mesenchyme-like compartment, over a 21-day culture period. We provide a step-by-step protocol for hydrogel preparation, cell seeding and culture conditions. The method is compatible with high-content imaging and quantitative analysis of organoid number, size distribution, and architectural complexity. This platform enables mechanistic studies of epithelial plasticity and environmental perturbations, providing a scalable and biologically relevant system for investigating early tissue-level changes associated with breast cancer risk.