Cell and Tissue Research

, Volume 375, Issue 2, pp 397–408 | Cite as

Enrichment of cancer stem cells by agarose multi-well dishes and 3D spheroid culture

  • Xiaoling Guo
  • Yong Chen
  • Weiping Ji
  • Xianwu Chen
  • Chao Li
  • Renshan GeEmail author
Regular Article


As the theory of cancer stem cells (CSCs) is maturing, CSC-targeted therapy is emerging as an important therapeutic strategy and seeking the ideal method for rapid enrichment and purification of CSCs has become crucial. So far, based on the known CSC phenotypes and biological characteristics, the methods for enrichment CSCs mainly include low adhesion culture, low oxygen culture, chemotherapy drug stimulation and side population (SP) sorting but these methods cannot realize quick enrichment of the desired CSCs. Herein, we adopt a novel method that efficiently enriches a certain amount of CSCs through agarose multi-well dishes using rubber micro-molds to make cancer cells into cell spheroids (3D). These 3D cancer cell spheroids in the proportions of expression of CSC biomarkers (single stain of CD44, CD44v6 and CD133 or double stain of both CD44 and CD133) were significantly higher than those of the conventional adherent culture (2D) using flow cytometry analysis. In addition, the expression levels of stemness transcription factors such as OCT4, NANOG and SOX2 in 3D were also significantly higher than that in 2D through Western blot (WB) and quantitative polymerase chain reaction (qPCR) assays. In addition, the CSCs in 3D could form colonies with different sizes in soft agar. In conclusion, we developed a new method to enrich some kinds of CSCs, which might be a benefit for future CSC-targeted therapy studies and anti-CSC drug screening applications.


Cancer stem cell 2D 3D Spheroid Enrichment 



This work was supported by the National Nature Science Foundation of China (81701426, 81730042 and 81771636), the Medical and Health Research Science and Technology Plan Project of Zhejiang Province (2018KY523, 2017KY473 and 2017ZB067) and the Public Welfare Science and Technology Plan Project of Wenzhou City (Y20170151, Y20160069 and ZS2017012).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Xiaoling Guo
    • 1
  • Yong Chen
    • 1
  • Weiping Ji
    • 2
  • Xianwu Chen
    • 1
  • Chao Li
    • 1
  • Renshan Ge
    • 1
    • 3
    Email author
  1. 1.Center of Scientific ResearchThe Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical UniversityWenzhouChina
  2. 2.Department of GastroenetrologyThe Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical UniversityWenzhouChina
  3. 3.Department of AnesthesiologyThe Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical UniversityWenzhouChina

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