Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Methods Mol Biol. 2022;2471:259-269. doi: 10.1007/978-1-0716-2193-6_14.
In the last decade, organoids became a tremendously popular technique in developmental and cancer biology for their high pathophysiological relevance to in vivo models with the advantage of easier manipulation, real-time observation, potential for high-throughput studies, and reduced ethical issues. Among other fundamental biological questions, mammary organoids have helped to reveal mechanisms of mammary epithelial morphogenesis, mammary stem cell potential, regulation of lineage specification, mechanisms of breast cancer invasion or resistance to therapy, and their regulation by stromal microenvironment. To exploit the potential of organoid technology to the fullest, together with optimal organoid culture protocols, visualization of organoid architecture and composition in high resolution in three dimensions (3D) is required. Whole-mount imaging of immunolabeled organoids enables preservation of the 3D cellular context, but conventional confocal microscopy of organoid cultures struggles with the large organoid sample size and relatively long distance from the objective to the organoid due to the 3D extracellular matrix (ECM) that surrounds the organoid. We have overcome these issues by physical separation of single organoids with their immediate stroma from the bulk ECM. Here we provide a detail protocol for the procedure, which entails single organoid collection and droplet-based staining and clearing to allow visualization of organoids in the greatest detail.
在过去的十年中，类器官在发育和癌症生物学领域成为一种非常流行的技术，因为它们与体内模型具有高度的生理相关性，具有易于操作、实时观察、高通量研究的潜力以及减少伦理问题等优势。在其他基本生物学问题中，乳腺类器官帮助揭示了乳腺上皮形态发生、乳腺干细胞潜能、谱系特化的调节、乳腺癌侵袭或对治疗的抵抗的机制，以及它们受基质微环境的调节。为了充分利用类器官技术的潜力，除了最佳的类器官培养方案外，还需要以高分辨率三维（3D）可视化类器官的结构和组成。免疫标记的类器官的整体成像可以保留 3D 细胞环境，但由于围绕类器官的 3D 细胞外基质（ECM），常规的共聚焦显微镜对类器官培养物的研究存在困难，因为类器官的样本量大，距离物镜较远。我们通过将单个类器官及其周围的基质与大块 ECM 进行物理分离来克服这些问题。这里我们提供了一个详细的方案，包括单个类器官的收集和基于液滴的染色和清除，以允许以最大的细节可视化类器官。
