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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.

Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy.