Mehta Pooja, Novak Caymen, Raghavan Shreya, Ward Maria, Mehta Geeta
Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI, 48109-2800, USA.
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48109-2800, USA.
Methods Mol Biol. 2018;1692:61-75. doi: 10.1007/978-1-4939-7401-6_6.
Cancer stem cells (CSC) are a vital component to the progression and reoccurrence of cancers, making them a primary target of study for both fundamental understanding of cancer biology and the development of effective and targeted treatments. CSCs reside in a complex 3D microenvironment, and the 3D spheroids are an indispensable tool in tumor biology due to their 3D structure and replication of the tumor microenvironment. Within this chapter the methodology for CSC isolation, suspension culture in hanging drop model, and characterization assays for CSC are described. First, the methodology for identifying and isolating CSCs from patient tumors, ascites, or cancer cell lines is described through the use of FACS analysis. Next, a detailed description of 3D hanging drop model for generating CSC spheroids is provided, followed by maintenance and monitoring techniques for extended 3D culture. Analysis methods are described for the quantification of CSC spheroid proliferation and viability tracking, throughout culture by on-plate alamarBlue fluorescence. Additional viability assays are described utilizing confocal microscopy with Live/Dead Viability/Cytotoxicity Kit. The characterization of CSCs populations within spheroids is described through FACS analysis. Further, an immunohistochemistry procedure is described for cell-cell and cell-matrix interaction assessment. Finally, several notes and tips for successful experiments with 3D CSC spheroids on the hanging drop model are provided. These methods are not only applicable to CSCs within a variety of tumor cell types, for not only understanding the fundamental tumor biology, but also for drug screening and development of preclinical chemotherapeutic strategies.
癌症干细胞（CSC）是癌症进展和复发的重要组成部分，这使得它们成为癌症生物学基础研究以及有效靶向治疗开发的主要研究对象。癌症干细胞存在于复杂的三维微环境中，而三维球体由于其三维结构以及对肿瘤微环境的模拟，成为肿瘤生物学中不可或缺的工具。在本章中，将描述癌症干细胞的分离方法、悬滴模型中的悬浮培养以及癌症干细胞的鉴定分析方法。首先，通过流式细胞术分析，描述从患者肿瘤、腹水或癌细胞系中识别和分离癌症干细胞的方法。接下来，详细介绍用于生成癌症干细胞球体的三维悬滴模型，随后介绍三维长期培养的维持和监测技术。描述了通过平板alamarBlue荧光在整个培养过程中对癌症干细胞球体增殖和活力追踪进行定量的分析方法。还介绍了利用共聚焦显微镜和活/死细胞活力/细胞毒性试剂盒进行的其他活力测定。通过流式细胞术分析描述球体中癌症干细胞群体的特征。此外，还描述了用于细胞间和细胞与基质相互作用评估的免疫组织化学程序。最后，提供了一些关于在悬滴模型上成功进行三维癌症干细胞球体实验的注意事项和提示。这些方法不仅适用于多种肿瘤细胞类型中的癌症干细胞，不仅可用于理解肿瘤生物学基础，还可用于药物筛选和临床前化疗策略的开发。
