Abstract Background Estrogen receptor α (ERα) signaling is a defining and driving event in most breast cancers; ERα is detected in malignant epithelial cells of 75% of all breast cancers (classified as ER-positive breast cancer) and, in these cases, ERα targeting is the main therapeutic strategy. However, the biological determinants of ERα heterogeneity and the mechanisms underlying therapeutic resistance are still elusive, hampered by the challenges in developing experimental models recapitulative of intra-tumoral heterogeneity and in which ERα signaling is sustained. Ex vivo cultures of human breast cancer tissue have been proposed to retain the original tissue architecture, epithelial and stromal cell components and ERα. However, loss of cellularity, viability and ERα expression are well-known culture-related phenomena. Methods BC samples were collected and brought to the laboratory. Then they were minced, enzymatically digested, entrapped in alginate and cultured for 1 month. The histological architecture, cellular composition and cell proliferation of tissue microstructures were assessed by immunohistochemistry. Cell viability was assessed by measurement of cell metabolic activity and histological evaluation. The presence of ERα was accessed by immunohistochemistry and RT-qPCR and its functionality evaluated by challenge with 17-β-estradiol and fulvestrant. Results We describe a strategy based on entrapment of breast cancer tissue microstructures in alginate capsules and their long-term culture under agitation, successfully applied to tissue obtained from 63 breast cancer patients. After 1 month in culture, the architectural features of the encapsulated tissue microstructures were similar to the original patient tumors: epithelial, stromal and endothelial compartments were maintained, with an average of 97% of cell viability compared to day 0. In ERα-positive cases, fibers of collagen, the main extracellular matrix component in vivo, were preserved. ERα expression was at least partially retained at gene and protein levels and response to ERα stimulation and inhibition was observed at the level of downstream targets, demonstrating active ER signaling. Conclusions The proposed model system is a new methodology to study ex vivo breast cancer biology, in particular ERα signaling. It is suitable for interrogating the long-term effects of anti-endocrine drugs in a set-up that closely resembles the original tumor microenvironment, with potential application in pre- and co-clinical assays of ERα-positive breast cancer.