Generation of Multicellular Breast Cancer Tumor Spheroids: Comparison of Different Protocols

J Mammary Gland Biol Neoplasia. 2016 Dec;21(3-4):89-98. doi: 10.1007/s10911-016-9359-2. Epub 2016 Aug 12.


Multicellular tumor spheroids are widely used models in tumor research. Because of their three dimensional organization they can simulate avascular tumor areas comprising proliferative and necrotic cells. Nonetheless, protocols for spheroid generation are still inconsistent. Therefore, in this study the breast cancer cell lines MCF-7, MDA-MB-231 and SK-BR-3 have been used to compare different spheroid generation models including hanging drop, liquid overlay and suspension culture techniques, each under several conditions. Experimental approaches differed in cell numbers (400-10,000), media and additives (25 % methocel, 25 % methocel plus 1 % Matrigel, 3.5 % Matrigel). In total, 42 different experimental setups have been tested. Generation of spheroids was evaluated by light microscopy and the structural composition was assessed immunohistochemically by means of Ki-67, cleaved poly (ADP-ribose) polymerase (cPARP) and mucin-1 (MUC-1) expression. Although the tested cell lines diverged widely in their capacity of forming spheroids we recommend hanging drops supplemented with 25 % methocel as the most reliable and efficient method with regard to success of generation of uniform spheroids, costs, experimental complexity and time expenditure in the different cell lines. MCF-7 cells formed spheroids under almost all analyzed conditions, and MDA-MB-231 cells under only one protocol (liquid overlay technique, 3.5 % Matrigel), while SK-BR-3 did not under neither condition. Therefore, we outline specific methods and recommend the use of adapted and standardized spheroid generation protocols for each cell line.

Keywords: 3D cultures; Breast cancer; MCF-7; MDA-MB-231; SK-BR-3; Spheroids; Tumor.

Publication types

  • Review
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology*
  • Cell Culture Techniques / methods
  • Cell Line, Tumor
  • Female
  • Humans
  • Ki-67 Antigen / metabolism
  • MCF-7 Cells
  • Mucin-1 / metabolism
  • Spheroids, Cellular / metabolism
  • Spheroids, Cellular / pathology*


  • Ki-67 Antigen
  • Mucin-1