title: 3D culture assays of murine mammary branching morphogenesis and epithelial invasion.
abstract: Epithelia are fundamental tissues that line cavities, glands, and outer body surfaces. We use three-dimensional (3D) embedded culture of primary murine mammary epithelial ducts, called "organoids," to recapitulate in days in culture epithelial programs that occur over weeks deep within the body. Modulating the composition of the extracellular matrix (ECM) allows us to model cell- and tissue-level behaviors observed in normal development, such as branching morphogenesis, and in cancer, such as invasion and dissemination. Here, we describe a collection of protocols for 3D culture of mammary organoids in different ECMs and for immunofluorescence staining of 3D culture samples and mammary gland tissue sections. We illustrate expected phenotypic outcomes of each assay and provide troubleshooting tips for commonly encountered technical problems.
authors.affiliation: Departments of Cell Biology and Oncology, Center for Cell Dynamics, Johns Hopkins School of Medicine, 855 N. Wolfe Street, 452 Rangos Building, Baltimore, MD, 21205, USA.
authors.forename: Kim-Vy
authors.initials: KV
authors.lastname: Nguyen-Ngoc
authors.forename: Eliah R
authors.initials: ER
authors.lastname: Shamir
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Huebner
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Beck
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Cheung
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Ewald
mesh_terms: D000818:Animals; D001681:Biological Assay; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation; D048430:Cell Shape / Q000187:drug effects; D003094:Collagen / Q000494:pharmacology; D024042:Collagen Type I / Q000494:pharmacology; D004338:Drug Combinations; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects; D005260:Female; D005455:Fluorescent Antibody Technique; D007797:Laminin / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*; D051379:Mice; D009024:Morphogenesis / Q000187:drug effects; D009940:Organoids / Q000166:cytology / Q000187:drug effects; D010641:Phenotype; D011509:Proteoglycans / Q000494:pharmacology; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D024042:Collagen Type I; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D012710:Serum Albumin, Bovine; D003094:Collagen