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Journal of Biomolecular Screening, Vol. 12, No. 7, 925-937 (2007)
DOI: 10.1177/1087057107306839

A Reliable Tool to Determine Cell Viability in Complex 3-D Culture: The Acid Phosphatase Assay

Juergen Friedrich

Institute of Pathology, University of Regensburg, Regensburg, Germany

Wolfgang Eder

Institute of Pathology, University of Regensburg, Regensburg, Germany

Juana Castaneda

Avalon Pharmaceuticals Inc., Germantown, Maryland

Markus Doss

Institute of Pathology, University of Regensburg, Regensburg, Germany

Elisabeth Huber

Institute of Pathology, University of Regensburg, Regensburg, Germany

Reinhard Ebner

Avalon Pharmaceuticals Inc., Germantown, Maryland

Leoni A. Kunz-Schughart

Institute of Pathology, University of Regensburg, Regensburg, Germany, OncoRay-Center for Radiation Research in Oncology, Faculty of Medicine Carl Gustav Carus, Dresden, Germany, leoni.kunz-schughart{at}oncoray.de

Cell-based assays are more complex than cell-free test systems but still reflect a highly artificial cellular environment. Incorporation of organotypic 3-dimensional (3-D) culture systems into mainstream drug development processes is increasingly discussed but severely limited by complex methodological requirements. The objective of this study was to explore a panel of standard assays to provide an easy-handling, standardized protocol for rapid routine analysis of cell survival in multicellular tumor spheroid-based antitumor drug testing. Spheroids of 2 colon carcinoma cell lines were characterized for evaluation. One of the assay systems tested could reliably be used to determine cell viability in spheroids. The authors verified that the acid phosphatase assay (APH) is applicable for single spheroids in 96-well plates, does not require spheroid dissociation, and is linear and highly sensitive for HT29 and HCT-116 spheroids up to diameters of 650 µm and 900 µm, consisting of 40,000 and 80,000 cells, respectively. Treatment of HT29 and HCT-116 cells with 5-fluorouracil, Irinotecan, and C-1311 revealed critically reduced drug efficacies in 3-D versus monolayer culture, which is discussed in light of literature data. The experimental protocol presented herein is a small but substantial contribution to the establishment of sophisticated 3-D in vitro systems in the antitumor drug screening scenario. (Journal of Biomolecular Screening 2007:925-937)

Key Words: cell-based assay • 3-D culture • spheroid • antitumor drug testing • cell viability


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