Abstract Worldwide, breast cancer (BC), is the most frequently diagnosed cancer among women. In the US, 1 out of 8 women may expect to be diagnosed with BC In their lifetime. Current models for studying BC tumor biology and BC drug discovery include 2D cultured cell lines and patient derived xenografts (PDX). As 2D cultured cells do not represent the 3D complexity of human tissue, and PDX models are time consuming and cost prohibitive, better tissue mimetic models are needed. Organoids are self-assembled 3D cultured cellular units that may mimic the corresponding human tissue structurally and functionally (1). Patient derived organoids (PDO) are developed from tissue of specific patients and may resemble the source tissue in molecular features, including recapitulation of patient specific responses to therapies (2). Here, we report development and characterization of novel breast PDO lines from fresh (≤24h post-surgery) and cryopreserved (≤7 days post-surgery) tissue. Breast tumor and normal, tumor adjacent (NTA) tissue were sourced from consented patients. Breast PDO were generated following modifications to a published protocol (3). Briefly, tissue samples were minced, digested with collagenase, and filtered. The filtrate was resuspended in Matrigel (Corning) and small drops were added on cell culture plates. Both tumor and NTA PDO were cultured using the same media and organoids were monitored using brightfield microscopy (Olympus CK40) and live cell imaging (Muvicyte, Perkin Elmer). PDO lines were analyzed for expression and distribution of important breast-specific biomarkers using confocal microscopy (ImagExpress, Molecular Devices). PDO lines displayed hormone receptors (ER, PR), cell surface (EpCAM, CD49f), cytoskeletal (cytokeratin 14, 19, E-cadherin, vimentin) and cell proliferation (Ki67) markers. Transcriptome profiles were analyzed using vendor services (Azenta). PDO were also tested for their susceptibility to breast cancer drugs, e.g., Tamoxifen, chlorambucil and Docetaxel. We have successfully generated one NTA ER+/PR+/HER2− PDO line and one tumor ER+/PR+/HER2− PDO line from different donors. Our breast PDO lines would be highly useful in understanding tumor biology, BC progression, signal transduction, and drug discovery/testing. Future efforts would include process scaling up and development of BC specific assays.