Generation of Mosaic Mammary Organoids by Differential Trypsinization
| Autor: | Oscar Cazares, Stefany Rubio, Lindsay Hinck, Hector Macias |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Tissue Fixation General Chemical Engineering Epithelium Extracellular matrix Tissue Culture Techniques Tissue culture Mice 0302 clinical medicine basal Psychology Trypsin mammary Mosaicism General Neuroscience Cell Differentiation Mammary Glands myoepithelial Cell biology Extracellular Matrix Organoids medicine.anatomical_structure 030220 oncology & carcinogenesis Female Cognitive Sciences Biotechnology 3D culture 1.1 Normal biological development and functioning organoid epithelial Biology General Biochemistry Genetics and Molecular Biology Article 03 medical and health sciences Mammary Glands Animal Issue 157 Underpinning research medicine Organoid Compartment (development) Animals breast luminal General Immunology and Microbiology Animal Myoepithelial cell Epithelial Cells Stem Cell Research Trypsinization 030104 developmental biology Biochemistry and Cell Biology Mammary gland morphogenesis Developmental Biology |
| Zdroj: | J Vis Exp |
| Popis: | Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient, and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional organoids that can be differentiated to produce milk over 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to culture in three-dimensions. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment. |
| Databáze: | OpenAIRE |
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