Wellcome - MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK.
Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
Methods Mol Biol. 2024;2805:3-18. doi: 10.1007/978-1-0716-3854-5_1.
Three-dimensional (3D) organoid cultures retain self-renewing stem cells that differentiate into multiple cell types that display spatial organization and functional key features, providing a highly physiological relevant system. Here we describe a strategy for the generation of 3D murine lung organoids derived from freshly isolated primary tracheal and distal lung epithelial stem cells. Isolated tracheas are subjected to enzymatic digestion to release the epithelial layer that is then dissociated into a single cell suspension for organoid culture. Lung epithelial cells are obtained from dissected lobes, which are applied to mechanical and enzymatic dissociation. After flow sorting, organoids are established from tracheal basal, secretory club, and alveolar type 2 cells in the defined conditioned medium that is required to sustain organoid growth and generate the differentiated cells. Multi-cell-type organoid co-culture replicates niches for distal epithelial stem cells to differentiate into bronchiolar and alveolar cell types. Established organoids can be fixed for wholemount staining and paraffin embedding, or passaged for further culture. Taken together, this protocol provides an efficient and validated approach to generate murine lung organoids, as well as a platform for further analysis.
三维（3D）类器官培养物保留自我更新的干细胞，这些干细胞可分化为多种具有空间组织和功能关键特征的细胞类型，提供了一个高度生理相关的系统。在这里，我们描述了一种从新鲜分离的原代气管和远端肺上皮干细胞生成 3D 鼠肺类器官的策略。分离的气管经过酶消化释放上皮层，然后将其解离成单细胞悬浮液用于类器官培养。从解剖的肺叶中获得肺上皮细胞，将其应用于机械和酶解离。经过流式细胞分选后，在定义的条件培养基中从气管基底细胞、分泌细胞和肺泡 II 型细胞中建立类器官，该培养基需要维持类器官生长并生成分化细胞。多细胞类型类器官共培养复制了远端上皮干细胞的龛位，使其分化为细支气管和肺泡细胞类型。已建立的类器官可用于固定进行全组织染色和石蜡包埋，或传代进行进一步培养。总之，该方案提供了一种高效且经过验证的生成鼠肺类器官的方法，以及进一步分析的平台。
