Figure 1. Multilineage and Luminal Mammary Organoids Can Be Generated Using MammoCult™ Organoid Kit (Human)

The human mammary epithelium comprises three main lineages: Hormone responsive (HR), secretory, and basal (myoepithelial), each maintained by its own population of lineage-restricted stem cells. HR cells have a strong luminal phenotype and express the estrogen receptor (ER) and the progesterone receptor (PR). Secretory lineage cells also reside within the luminal layer but display an intermediate phenotype between HR and basal cells; during pregnancy, they proliferate to generate alveolar daughter cells that synthesize and secrete milk during lactation. The MammoCult™ Organoid Kit (Human) enables selective culture of these distinct epithelial stem cell populations by combining specific basal media with defined supplements. (A) Large, branched, multilineage organoids can be generated by embedding epithelial fragments and aggregates that contain both luminal and basal cells in Matrigel®/collagen I domes and culturing them in MammoCult™ Multilineage Organoid Medium (MOM; Basal Medium 1 with Supplements A - D). To promote the formation of a lumen and lobules, the medium is switched to a growth factor-reduced (GFR) formulation (Basal Medium 2 with Supplement A) on Days 10 - 12. (B) To generate luminal-enriched, cystic organoids, cells are embedded in Matrigel® domes and cultured in MammoCult™ Luminal Organoid Medium (LOM; Basal Medium 2 with Supplements A - C). By Days 8 - 18, organoids have a cystic morphology and are ready for passaging during this period. Basal cells are not selected to grow in LOM, eliminating the need to sort luminal cells prior to initiating the cultures. To expand and stabilize cell samples prior to organoid culture, pre-culturing in EpiCult™ Plus or LOM is recommended to improve performance. This modular system supports targeted studies of human mammary epithelial biology. Scale bars = 100 μm.

Figure 2. MammoCult™ Luminal Organoid Medium Enables the Generation of Pure Luminal Organoids that Preserve Key Functional Markers

Non-sorted primary human mammary epithelial cells seeded in MammoCult™ Luminal Organoid Medium (LOM) generated pure luminal organoids that recapitulate key structural, lineage, and functional features of the human mammary epithelium. (A) Brightfield imaging shows organoids display characteristic cystic morphology consistent with luminal lineage enrichment. (B - H) Comprehensive immunostaining and FACS analyses confirm that these organoids are composed of keratin 8+, keratin 14- luminal epithelial cells, with limited basal cell contamination. Importantly, these cultures preserve both major luminal cell types: hormone-sensing and secretory populations, validated by the expression of (C,E,G) MUC1, ER, PR, and KIT. The ability to generate ER⁺ and PR⁺ hormone-sensing luminal cells, historically considered very challenging to culture, highlights a key functional advantage of MammoCult™ LOM. (D) Apical localization of MUC1 further demonstrates correct epithelial polarity as it appears in vivo. (F) Additionally, the detection of rare K8+/K14+ double-positive luminal cells underscores the model’s relevance for studying disease-associated cell states, including those implicated in basal-like breast cancer. (H) Functional potential of the luminal organoids is supported by the presence of PMCA2, a calcium pump associated with lactation, suggesting that these organoids might retain the potential functional capacity to lactate. Together, these findings demonstrate that organoids grown in MammoCult™ LOM are luminal lineage-enriched and recapitulate key features of the luminal compartment within the human mammary gland, thereby providing a robust, previously unattainable platform for studying luminal biology and disease mechanisms. Scale bars = 100 µm.

Figure 3. MammoCult™ Multilineage Organoid Medium Supports Flexible Generation of Lineage-Restricted and Multilineage Organoids

(A) Organoids generated from single-cell suspensions cultured in MammoCult™ Multilineage Organoid Medium (MOM) generate predominantly lineage-restricted structures that include pure luminal or pure basal organoids. Organoids were stained for proliferation marker Ki-67, epithelial cell adhesion molecule (EpCAM), secretory markers K14 (basal) and K8 (luminal), and DAPI. When transferred to Growth Factor Reduced (GFR) Medium, proliferation decreases and basal-derived organoids undergo morphological differentiation, forming lumens (asterisk) and lobular structures (n = 10). (B) Organoids derived from dissociated breast tissue fragments cultured in MammoCult™ MOM generate polarized multilineage structures composed of an inner layer of K8-expressing luminal cells, an outer layer of smooth muscle actin (SMA), and K14-expressing basal cells (n = 5). These results demonstrate that MammoCult™ MOM enables flexible control of organoid composition, supporting either lineage-restricted or multilineage models depending on the starting material, allowing researchers to tailor cultures to specific biological questions or to better recapitulate native tissue complexity. Scale bars = 100 μm.

Figure 4. MammoCult™ Organoid Kit (Human) Supports the Growth of TNBC and Luminal Breast Cancer Organoids

Representative light microscopy images show organoids derived from (A) triple-negative breast cancer (TNBC) and (B, C) two Luminal A tumor organoid lines that were cultured in MammoCult™ Luminal Organoid Medium (LOM). (D) Organoids derived from undefined HUB-01-B2-186 tumor organoid line cultured in MammoCult™ Multilineage Organoid Medium (MOM). Flow cytometry analysis of EpCAM versus CD49f confirms that (E) TNBC organoids are composed predominantly of basal-like cells, while (F) the HUB-01-B2-125 Luminal A organoid line is composed of predominantly luminal cells, consistent with their respective tumor origins. These results suggest that MammoCult™Organoid Kit (Human) supports the culture of breast cancer organoid lines enabling disease modeling of TNBC and luminal breast cancer. Scale bars = 100 μm.

Figure 5. MammoCult™ Multilineage Organoid Medium Supports Physiologically Relevant Branching Morphogenesis Through Fibroblasts and Collagen I

(A) Human mammary epithelial cells were sorted into three lineages and recombined into aggregates using AggreWellTM400 Microwell 24-well Culture Plates. After 6 days in MammoCult™Multilineage Organoid Medium (MOM), aggregates were stained for K8, K14, and fibroblast specific protein 1 (FSP1). Upon transfer to organoid culture, aggregates containing fibroblasts displayed increased branching and ductal elongation compared to aggregates lacking fibroblasts (n = 3). (B) Non-sorted mammary epithelial cells were seeded into 3D organoid culture in Matrigel® domes supplemented with increasing concentrations of collagen I (n = 3). Organoid branching was directly proportional to the concentration of collagen I, but declined after subsequent passaging. These results demonstrate that branching morphogenesis in mammary organoids can be initiated by either fibroblasts or collagen I; however, sustained growth and branching during passaging require the addition of fibroblasts in cultures supplemented with collagen I at an ideal concentration of 2 mg/ml. Scale bars = 100 μm.

Figure 6. MammoCult™ Organoid Kit (Human) Supports the Growth of Luminal Breast Epithelial Cells from Post-Menopausal Donors

Brightfield images of organoids generated from flow-purified (A) hormone-responsive and (B) secretory lineage cells isolated from normal breast tissue of a post-menopausal donor. Cells were cultured in MammoCult™ Luminal Organoid Medium (LOM) for 10 days, forming distinct, polarized, lumen-containing structures, demonstrating support for luminal epithelial cell growth from post-menopausal tissue. Scale bar = 400 µm. Images courtesy of Drs. Martin Hirst and Shrinka Sen, Michael Smith Laboratories, University of British Columbia.

Figure 7. MammoCult™ Organoid Kit (Human) Supports a 2D to 3D Workflow While Preserving Key Lineage Markers

(Left) Human primary mammary epithelial cells are grown in two-dimensional (2D) adherent cell culture to passage 2 using either EpiCult™ Plus or MammoCult™ Luminal Organoid Medium (LOM). Pre-culturing cells in EpiCult™ Plus generated a mixture of K8- expressing luminal cells and K14-expressing basal cells (n = 4). Flow cytometry analysis of EpCAM versus CD49f confirms the presence of the luminal and basal cell populations. In contrast, culture in MammoCult™ LOM selectively expanded K8-expressing luminal cells, which formed tightly packed clusters (n = 6). Flow cytometry analysis revealed a predominantly luminal phenotype (EpCAM⁺/CD49f⁺, 92.77%), with minimal basal cell presence (4.59%). These luminal cells also expressed estrogen receptor (ER), progesterone receptor (PR), and MUC1, confirming their lineage identity. (Right) Human breast tissue samples can also be seeded directly into 3D organoid culture, otherwise pre-culturing cells primes them for organoid seeding, enhancing organoid-forming potential and enabling a seamless transition from 2D to 3D culture. Cells grown in EpiCult™ Plus were transferred into MammoCult™ Multilineage Organoid Medium (MOM), while LOM culture remained in the same medium. Light and fluorescence microscopy images reveal distinct morphologies in the resulting organoids that reflect their lineage of origin: basal cells generate branched structures, while luminal cell-derived organoids form hollow spherical structures (n = 4). Immunocytochemistry was performed to detect luminal markers (K8) and basal markers (K14, P63), demonstrating how MammoCult™ Organoid Kit (Human) supports a complete 2D-to-3D culture workflow for human mammary epithelial cells, while preserving key functional markers. Scale bars = 100 µm.