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

The mouse mammary epithelium comprises two main lineages: luminal and basal (myoepithelial), each maintained by its own population of lineage-restricted stem cells. The luminal lineage can be further subdivided into 2 sublineages: hormone responsive (HR) and secretory. HR cells have a strong luminal phenotype and express the estrogen receptor (ER) and the progesterone receptor (PR). In contrast, secretory lineage cells display low expression of ER and PR; during pregnancy, they proliferate to generate alveolar daughter cells that synthesize and secrete milk during lactation. The MammoCult™ Organoid Kit (Mouse) 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 mouse mammary epithelial fragments and aggregates in Matrigel®/collagen I domes and culturing them in MammoCult™ Multilineage Organoid Medium (MOM; Basal Medium with Supplements A + B). (B) To generate luminal-enriched, cystic organoids, cells are embedded in Matrigel® domes and cultured in MammoCult™ Luminal Organoid Medium (LOM; Basal Medium with Supplement A). By Days 10 - 13, 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. This modular system supports targeted studies of mouse mammary epithelial biology. Scale bars = 500 μm.

Figure 2. MammoCult™ Organoid Kit (Mouse) Generates Mammary Organoids with Physiologically Relevant Luminal and Basal Architecture

(A) Mature multilineage organoids generated using Multilineage Organoid Medium express both luminal and basal mammary epithelial lineages. Luminal cells are identified by the expression of keratin 8 (K8), estrogen receptor (ER), and progesterone receptor (PR), while basal cells express keratin 14 (K14) and smooth muscle actin (SMA). Both K14 and SMA are localized to the outer layer of the organoid, while K8-expressing cells are positioned on the inside. This spatial organization mirrors the in vivo mammary epithelium where contractile cells encase luminal secretory cells to facilitate milk ejection. (B) In contrast, organoids cultured in Luminal Organoid Medium show expression of K8 and ER, consistent with a pure luminal phenotype, and absence of basal cell markers. Together, these data demonstrate that the MammoCult™ Organoid Kit (Mouse) supports the generation of mammary organoids that faithfully reproduce both the cellular composition and structural organization of the in vivo mammary epithelium across passages. Data was used with permission from Mona Shehata, University of Cambridge.

Figure 3. Mammary Organoids Cultured in MammoCult™ Organoid Kit (Mouse) Exhibit Secretory Differentiation and Lactation-Associated Gene Expression

(A) Multilineage mammary organoids were dissociated and stained to detect ALDEFLUORBRIGHT cells, a phenotype associated with secretory lineage cells. (B) Live cell imaging confirmed the presence of ALDEFLUORBRIGHT cells within multilineage organoids. Scale bars = 100 μm. (C) To assess the functional relevance of this secretory population, pre-established mammary organoids (Passage 1, Day 10) were treated for 4 days with lactogenic hormones: 5 µg/mL prolactin, 5 µg/mL insulin, and 100 ng/mL hydrocortisone. This treatment upregulated the transcripts associated with milk production, including casein 2 (Csn2), lactoferrin (Ltf), and whey acidic protein (Wap). Expression of transcripts for casein alpha s1 (Csn1s1) were not significantly upregulated. Gene expression data was normalized to untreated cells from the same mouse (n = 4), and statistical significance was determined using paired t-tests. These results demonstrate that mammary organoids cultured using the MammoCult™ Organoid Kit (Mouse) exhibit key lactation-associated genes in response to hormonal cues, supporting their use as physiologically relevant in vitro models of mammary gland function.

Figure 4. MammoCult™ Organoid Kit (Mouse) Is Compatible with Multiple Mouse Strains

Brightfield and immunocytochemistry (ICC) images of multilineage organoids cultured using MammoCult™ Organoid Kit (Mouse) from four different mouse strains: C57BL/6J, CD-1, FVB, and Balb C. Images were captured on Day 9 of Passage 2. Organoids from all strains exhibit strong growth and branching morphology with distinct epithelial lineages as indicated by ICC staining of luminal marker K8 (green) and basal cell marker K14 (red). These results demonstrate the compatibility of MammoCult™ Organoid Kit (Mouse) across diverse sample sources.

Figure 5. MammoCult™ Multilineage Organoid Medium (Mouse) Supports Robust Short-Term Expansion of Mammary Organoids from Single-Cell Suspensions

Freshly dissociated mouse mammary epithelial cells were seeded as a single-cell suspension and cultured in MammoCult™ Multilineage Organoid Medium (Mouse). By Day 10, large branched organoids dominate the culture (foreground), while smaller cystic luminal organoids are also present (background). Although passaging organoids as single cells leads to rapid expansion, these cultures may undergo early senescence. For long-term multilineage culture, organoids should be passaged as fragments. This medium supports the expansion and differentiation of multiple mammary epithelial lineages in a single culture system. For long-term maintenance of luminal organoids, Luminal Organoid Medium is recommended. Scale bar = 250 μm.

Figure 6. MammoCult™ Multilineage Organoid Medium (Mouse) Supports Long-Term Maintenance of Genomically Stable Mammary Organoids When Passaged As Tissue Fragments

(A) Growth curves of mouse mammary organoids when passaged as fragments and single cells in MammoCult™ Multilineage Organoid Medium (Mouse). Although passaging organoids as single cells leads to rapid initial expansion, these cultures undergo early senescence and develop karyotypic abnormalities. (B) Brightfield images of multilineage organoids on Day 14 of passage 0 (P0D14), passage 5 (P5D14), and passage 10 (P10D14), and the preserved normal karyotype of P10D14 organoids when passaged as small fragments. Together, these results demonstrate that MammoCult™ Multilineage Organoid Medium (Mouse) supports robust long-term growth of mammary organoids while maintaining genomic stability when cultures are passaged as fragments. Scale bar = 500 μm.

Figure 7. Expansion in EpiCult™ Plus Preserves Organoid-Forming Potential for Mammary Organoid Culture Using MammoCult™ Organoid Kit (Mouse)

(A) Phase contrast image of mouse mammary epithelial cells at passage 2, cultured in 2D in EpiCult™ Plus medium. The culture contains both (B) K8-positive luminal cells and K14-positive basal cells. Following expansion in EpiCult™ Plus, cells were harvested and seeded into organoid assays using Multilineage Organoid Medium (MOM). (C) Day 7 organoid culture displays a range of morphologies, including branching structures and cystic luminal formations. (D) Multilineage organoids show an inner layer of K8-expressing luminal cells and an outer layer of K14-expressing basal cells. (E) Luminal cell organoids consist exclusively of K8-expressing luminal cells, many of which express (F) estrogen receptor (ER). Pre-culturing in EpiCult™ Plus primes cells for organoid seeding, helping enhance organoid-forming potential while supporting a seamless transition from 2D to 3D culture workflows.