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The NG organoid model was initially developed by the Gupta Lab at MIT and refined through a collaboration with the Kuperwasser Lab at Tufts. The Gupta and Kuperwasser labs later merged into a joint laboratory, with Dr. Rauner continuing to advance, refine, and utilize the model as a postdoctoral fellow and within her independent lab.

3 pillars make NG breast organoids unique compared with previous models:


I. Biomimetic extracellular matrix (ECM): Many other models rely on reconstituted basement membranes (such as Matrigel), collagen or a combination of the two. The NG organoid model offers a more physiologically relevant ECM composition. It includes, in addition to collagen, essential components of the breast stroma: laminins, fibronectins, and hyaluronic acid. These components play a key role in proper functioning of breast epithelial cells, and in breast cancer progression.


II. Defined Culture Media: NG breast organoids do not require chemical inhibitors or animal serum. This eliminates potential confounding factors and provides a more controlled experimental environment for drug testing.


III. Patient-Derived Organoids: Unlike models that utilize mouse primary cells, human cell lines, or induced pluripotent stem cells (iPSCs), NG organoids originate from patient-derived single cells. This approach ensures a more accurate representation of human biology and enables the study of individual variations and responses.


NG breast organoids recapitulate key aspects of the human breast tissue:


Breast Terminal Ductal-Lobular Unit (TDLU): Many organoid models of the breast yielded spherical structures with or without budding. NG breast organoids have complex ducal-lobular architecture that closely mimics the breast's TDLU.


Cellular heterogeneity and gene expression: Side-by-side single cell RNA-seq analysis of normal breast cells and NG breast organoids reveals that NG breast organoids reliably recapitulate the main epithelial cell populations in the breast. This crucial evidence indicates the donor-derived single-cell that forms the organoid undergoes a bona fide process of cell differentiation that recapitulates breast tissue development.


Hormone responsiveness: NG breast organoids express estrogen and progesterone receptors, and respond to hormones by expanding and hollowing alveoli, and to prolactin by secreting lipid droplets.

NG breast organoids can be genetically modified, adapted for high-throughput and tracked with live-imaging:


Transduced NG organoids stably express desired genetic constructs, allowing for a targeted study of breast tissue function and behavior. This is especially vital for understanding normal mammary gland development, tissue regeneration, disease pathogenesis, and drug responses.













Genetic reporters can be employed to visualize and track specific cellular events in real-time, such as gene expression, protein localization, and cellular differentiation.


Introducing genetic mutations that mimics disease conditions allows for the study of disease onset, progression, and potential therapeutic interventions, making it highly relevant for breast cancer research and other breast-related conditions.


CRISPR-Cas9 system combined with organoid technology can lead to high-throughput genetic screens to understand gene function, disease resistance mechanisms, and potential drug targets.


High-throughput organoid assays can serve as a model to test drug efficacy and toxicity, especially when developed from patient-derived cells. This holds promise for personalized medicine where treatments are tailored to individual patients.

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Next-Generation Organoid Technology

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