title: Generation of Multicellular Breast Cancer Tumor Spheroids: Comparison of Different Protocols.
abstract: 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.
authors: Department of Obstetrics, Placenta-Lab, Bachstraße 18, 07743, Jena, Germany.
authors: Karolin
authors: K
authors: Froehlich
authors: Department of Anatomy, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173, Hannover, Germany.
authors: Jan-Dirk
authors: JD
authors: Haeger
authors: Department of Obstetrics, Placenta-Lab, Bachstraße 18, 07743, Jena, Germany.
authors: Julia
authors: J
authors: Heger
authors: Department of Obstetrics, Placenta-Lab, Bachstraße 18, 07743, Jena, Germany.
authors: Jana
authors: J
authors: Pastuschek
authors: Department of Obstetrics, Placenta-Lab, Bachstraße 18, 07743, Jena, Germany.
authors: Stella Mary
authors: SM
authors: Photini
authors: Department of Obstetrics, Placenta-Lab, Bachstraße 18, 07743, Jena, Germany.
authors: Yan
authors: Y
authors: Yan
authors: Institute of Pharmacology and Toxicology, University Hospital Jena, Drackendorfer Straße 1, 07747, Jena, Germany.
authors: Amelie
authors: A
authors: Lupp
authors: Department of Anatomy, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173, Hannover, Germany.
authors: Christiane
authors: C
authors: Pfarrer
authors: KIMIII Department of Experimental Nephrology, University Hospital Jena, 07743, Jena, Germany.
authors: Ralf
authors: R
authors: Mrowka
authors: Department of Obstetrics, Placenta-Lab, Bachstraße 18, 07743, Jena, Germany.
authors: Ekkehard
authors: E
authors: Schleußner
authors: Department of Obstetrics, Placenta-Lab, Bachstraße 18, 07743, Jena, Germany. markert@med.uni-jena.de.
authors: Udo R
authors: UR
authors: Markert
authors: Department of Obstetrics, Placenta-Lab, Bachstraße 18, 07743, Jena, Germany.
authors: André
authors: A
authors: Schmidt
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D018929:Cell Culture Techniques / Q000379:methods; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D019394:Ki-67 Antigen / Q000378:metabolism; D061986:MCF-7 Cells; D018396:Mucin-1 / Q000378:metabolism; D018874:Spheroids, Cellular / Q000378:metabolism / Q000473:pathology*
chemical_list: D019394:Ki-67 Antigen; D018396:Mucin-1
keywords: 3D cultures; Breast cancer; MCF-7; MDA-MB-231; SK-BR-3; Spheroids; Tumor