Among females, breast cancer is the most diagnosed cancer, accounting for almost 1-in-4 cancer cases, and the leading cause of cancer-related deaths. In the twenty-first century, cancer is expected to rank as the leading cause of death worldwide, and the single most important barrier to increasing life expectancy (GLOBOCAN2018) (Ferlay et al., 2019). This integrative 3D model stands out as a promising in vitro platform for studying breast stroma-parenchyma interactions, both under physiological and pathological settings. In particular, we show that retrieved cell populations could be discriminated by flow cytometry using cell-type specific markers. Furthermore, the hybrid system could be easily dissolved for cell recovery and subsequent analysis by standard cellular/molecular assays. This generated a heterotypic 3D model that partially recreates both stromal and parenchymal compartments of breast tissue, promoting cell-cell and cell-matrix crosstalk. Throughout time, epithelial cells formed prototypical mammary acini-like structures, in close proximity with fibroblasts and their ECM. On a second stage, a peptide-modified alginate pre-gel laden with mammary gland epithelial cells was used to fill the scaffold's pores, forming a hydrogel in situ by ionic crosslinking. Seeded fibroblasts were able to adhere, spread and produce endogenous ECM, providing adequate coverage of the scaffold surface, without obstructing the pores. To build the model, porous scaffolds were produced by extrusion 3D printing of peptide-modified alginate hydrogels, and then populated with human mammary fibroblasts. Here, we describe an advanced 3D in vitro model of breast tissue, combining multiple components, namely stromal cells and their extracellular matrix (ECM), as well as parenchymal epithelial cells, in a hybrid system. Improved knowledge on the crosstalk between parenchymal and stromal mammary cells should provide important insights on breast tissue dynamics, both under healthy and diseased states. Breast tissue consists of an epithelial parenchyma embedded in stroma, of heterogeneous and complex composition, undergoing several morphological and functional alterations throughout females' lifespan.

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