Self-Renewal and CSCs In Vitro Enrichment: Growth as Floating Spheres
Open accessAbstract
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.
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CSC populations can be cultured to generate spheroids that retain stem-like properties, including self-renewal and resistance to standard chemotherapeutics.
“These CSC populations can then be cultured to generate spheroids that retain stem-like properties, including self-renewal and resistance to standard chemotherapeutics [219].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [219]
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Research works printed but not linked: 22
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Shah, S., & D’Souza, G. G. M. (2025) · Cells · doi:10.3390/cells14100732 · Cited by 66 · see citing works
“These CSC populations can then be cultured to generate spheroids that retain stem-like properties, including self-renewal and resistance to standard chemotherapeutics [219].”
- Authors
- Mehta, P., Novak, C., Raghavan, S., Ward, M., & Mehta, G.
- Affiliations
- University of Michigan (US)
- Corresponding author
- Mehta, Geeta
- Venue
- Methods in Molecular Biology
- Publisher
- Springer New York
- Published
- 2017-10-07
- Volume
- 1692
- Pages
- 61-75
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- PMC
- PMC5925737
- OpenAlex
- W2761057799
- Cited by
- 43
- References
- 0
- Citations by year
- 2026: 3 · 2025: 3 · 2024: 5 · 2023: 2 · 2022: 7 · 2021: 7 · 2020: 9 · 2019: 4 · 2018: 3
- FWCI
- 11.69
- Type
- book chapter
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of Dental and Craniofacial Research (T32DE007057)
- Topics
- Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
- Health Sciences › Medicine › Genetics › Mesenchymal stem cell research
- Physical Sciences › Mathematics › Modeling and Simulation › Mathematical Biology Tumor Growth
- Keywords
- cancer stem cells, multicellular spheroids, hanging drop culture, self-renewal, immunohistochemistry, cell-matrix interactions, tumor microenvironment, drug screening
- MeSH terms
- Cell Survival, Female, Humans, Ovarian Neoplasms, Neoplastic Stem Cells, Spheroids, Cellular, Cell Line, Tumor
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