title: A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay.
abstract: 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 microm and 900 microm, 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.
authors.affiliation: Institute of Pathology, University of Regensburg, Regensburg, Germany.
authors.forename: Juergen
authors.initials: J
authors.lastname: Friedrich
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Eder
authors.forename: Juana
authors.initials: J
authors.lastname: Castaneda
authors.forename: Markus
authors.initials: M
authors.lastname: Doss
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Huber
authors.forename: Reinhard
authors.initials: R
authors.lastname: Ebner
authors.forename: Leoni A
authors.initials: LA
authors.lastname: Kunz-Schughart
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D003110:Colonic Neoplasms / Q000201:enzymology / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D015203:Reproducibility of Results
chemical_list: D000135:Acid Phosphatase