# US 2025/0154472 A1 · Culture Medium for Expanding Breast Epithelial Stem Cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0154472 A1 - Document ID: US20250154472A1 - Status: Published application - Kind: A1 - Application number: 18/753,207 - Filed: 2024-06-25 - Published: 2025-05-15 - Earliest claimed: 2014-11-27 - Assignees: Koninklijke Nederlandse Akademie van Wetenschappen - Applicants: Koninklijke Nederlandse Akademie Van Wetenschappen - Inventors: Lars Norman Sachs; Ana Gracanin; Jarno Drost - Web page: https://patentgrep.com/patent/US20250154472A1 ## CPC - C12N 5/0695: {Stem cells; Progenitor cells; Precursor cells} - C12N 5/0631: {Mammary cells} - C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00) - C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2) - C12N 2501/415: Wnt; Frizzeled - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The invention relates to improved culture methods for expanding epithelial stem cells and obtaining organoids, to culture media involved in said methods, and to uses of said organoids. ## Claims ### Claim at position 1 -30. (canceled) ### Claim 31 A breast cancer organoid comprising a population of breast epithelial stem cells obtained from primary breast cancer or metastatic breast cancer, wherein the breast cancer organoid has a central lumen, and wherein the breast cancer organoid has been cultured or can be cultured for more than 6 passages. ### Claim 32 (depends on 31) The breast cancer organoid of claim 31, wherein the breast cancer organoid is in a culture medium comprising an ErbB3/4 ligand, one or more FGFR2b ligands, and a Wnt agonist, wherein the Wnt agonist is an Lgr5 agonist or Wnt3a. ### Claim 33 (depends on 31) The breast cancer organoid of claim 31, wherein the breast cancer organoid has been cultured for at least 2 months, and wherein the breast epithelial stem cells have been passaged approximately every 1 to 4 weeks. ### Claim 34 (depends on 31) The breast cancer organoid of claim 31, wherein the breast organoid has a cyst like structure with a central lumen. ### Claim 35 (depends on 34) The breast cancer organoid of claim 34, wherein the central lumen of the cyst like structure: (a) does not comprise any cells; or (b) comprises breast epithelial stem cells. ### Claim 36 (depends on 34) The breast cancer organoid of claim 34, wherein the breast epithelial cells surround the central lumen. ### Claim 37 (depends on 31) The breast cancer organoid of claim 31, comprising: (a) a continuous epithelium and intercellular adhesion; and/or (b) luminal cells but no basal cells. ### Claim 38 (depends on 31) The breast cancer organoid of claim 31, wherein the breast epithelial stem cells are multipotent, oligopotent, or unipotent organ specific adult stem cells. ### Claim 39 (depends on 31) The breast cancer organoid of claim 31, wherein the breast cancer organoid consists essentially of epithelial cells. ### Claim 40 (depends on 31) The breast cancer organoid of claim 31, wherein non-epithelial stem cells are absent from the breast cancer organoid. ### Claim 41 (depends on 31) The breast cancer organoid of claim 31, wherein the breast cancer organoid: (a) comprises breast epithelial stem cells which are able to actively divide; (b) has a section which is formed of multiple layers; and/or (c) has a monolayer of pseudo stratified cells folds so that it encloses lumina between the folds. ### Claim 42 (depends on 41) The breast cancer organoid of claim 41, wherein the breast epithelial stem cells are able to differentiate to all major differentiated cell lineages present in the corresponding in vivo tissue. ### Claim 43 (depends on 32) The breast cancer organoid of claim 32, wherein: (a) the ErbB3/4 ligand is a neuregulin polypeptide; (b) the one or more FGFR2b ligands is FGF7 and/or FGF10; and/or (c) the Wnt agonist is an Lgr5 agonist. ### Claim 44 (depends on 43) The breast cancer organoid of claim 43, wherein the neuregulin polypeptide is encoded by NRG1 or comprises the amino acid sequence of SEQ ID NO: 27. ### Claim 45 (depends on 43) The breast cancer organoid of claim 43, wherein the Lgr5 agonist is a Rspondin or a Rspondin mimic. ### Claim 46 (depends on 32) The breast cancer organoid of claim 32, wherein the culture medium further comprises: (a) a BMP inhibitor; (b) one or more components selected from: B27, N acetylcysteine, Nicotinamide, a ROCK inhibitor, a TGF beta inhibitor, and a p38 inhibitor; or (c) B27, N acetylcysteine, Nicotinamide, a ROCK inhibitor, a TGF beta inhibitor, and a p38 inhibitor. ### Claim 47 (depends on 32) The breast cancer organoid of claim 32, wherein: (a) the BMP inhibitor is Noggin; (b) the ROCK inhibitor is Y 27632; (c) the TGF beta inhibitor is A83 01; and/or (d) the p38 inhibitor is SB 202190. ### Claim 48 (depends on 32) The breast cancer organoid of claim 32, wherein the culture medium further comprises one or more components selected from: one or more further receptor tyrosine kinase ligands and a p53 stabilizing agent. ### Claim 49 (depends on 48) The breast cancer organoid of claim 48, wherein: (a) the one or more further receptor tyrosine kinase ligands are EGF, PDGF and/or amphiregulin; and/or (b) the p53 stabilizing agent is a member of the Nutlin family. ### Claim 50 (depends on 49) The breast cancer organoid of claim 49, wherein: (a) the PDGF is PDGF CC; and/or (b) the member of the Nutlin family is Nutlin 3. ### Claim 51 (depends on 31) The breast cancer organoid of claim 31 obtained by a method comprising: providing a population of breast epithelial stem cells obtained from primary breast cancer or metastatic breast cancer; providing a culture medium comprising an ErbB3/4 ligand, one or more FGFR2b ligands and a Wnt agonist, wherein the Wnt agonist is an Lgr5 agonist or Wnt3a; and wherein the culture medium is suitable for expanding the population of breast epithelial stem cells for at least 6 passages; contacting the breast epithelial stem cells with the culture medium; and culturing the breast epithelial stem cells under appropriate conditions to form a breast cancer organoid. ### Claim 52 (depends on 51) The breast cancer organoid of claim 51, wherein the ErbB3/4 ligand is a neuregulin polypeptide. ### Claim 53 (depends on 51) The breast cancer organoid of claim 51, wherein the culture medium comprises an ErbB3/4 ligand, one or more FGFR2b ligands, and a Wnt agonist, wherein the Wnt agonist is an Lgr5 agonist or Wnt3a, and wherein the ErbB3/4 ligand is a neuregulin polypeptide encoded by NRG1. ### Claim 54 (depends on 31) A composition comprising the breast cancer organoid of claim 31. ### Claim 55 (depends on 31) A method for a drug discovery screen; toxicity assay; research of tissue embryology, cell lineages, or differentiation pathways; gene expression study including recombinant gene expression; research of mechanisms involved in tissue injury or repair; research of inflammatory and infectious diseases; studies of pathogenetic mechanisms; or study of mechanisms of cell transformation or aetiology of cancer, comprising utilizing the breast cancer organoid of claim 31. ### Claim 56 (depends on 31) A method of treating a disease in a subject, comprising administering an effective amount of the breast cancer organoid of claim 31, or cells derived therefrom to a subject in need thereof. ### Claim 57 A breast organoid comprising a population of breast epithelial stem cells in a culture medium comprising an ErbB3/4 ligand. ### Claim 58 (depends on 57) The breast organoid of claim 57, wherein the culture medium further comprises one or more FGFR2b ligands, and a Wnt agonist, wherein the Wnt agonist is an Lgr5 agonist or Wnt3a.