The multilayered postconfluent cell culture as a model for drug screening

New drug development requires simple in vitro models that resemble the in v ivo situation more in order to select active drugs against solid tumours an d to decrease the use of experimental animals. In this paper, we review the characteristics and scope of a relatively simple cell-culture system with a three-dimensional organisation pattern - the multilayered postconfluent c ell culture model. Solid tumour cell lines from diverse origins when grown in V-bottomed microtiter plates reach confluence in 3-5 days and then start to form multilayers. The initial exponential growth of the culture is foll owed by a plateau phase when cells reach confluence. This produces changes in the morphology of the cells. For some cell lines, it is possible to obse rve cell differentiation. A substantial advantage of the system is the use of the sulforodamine B (SRB) assay to determine relative cell growth or via bility, which allows semiautomation of the experiments. Several experiments were performed to assess the differences and similarities between cells cu ltured as monolayers and multilayers, and eventually, compared with the res ults for solid tumours and some other models such as spheroids. Cell-cycle analysis for multilayers showed a lower S-phase arrest, which is accompanie d by a decrease in the expression of cell-cycle-related proteins and a decr ease in cellular nucleotide pools. Gene and protein expression of topoisome rase I, topoisomerase II and thymidylate synthase expression were lower for multilayers, but no substantial changes were observed for the expression o f DT-diaphorase. P53 expression increased. Multilayer cultures present dist inctive properties for drug transport across the membrane, drug accumulatio n and retention. In fact, the transport of antifolates across the membrane, accumulation of topotecan and gemcitabine-triphosphate are reduced in mult ilayers when compared with monolayers, which may be related to a decrease i n drug penetration to the inner regions of the multilayers. Alteration of t hese pharmacodynamic parameters is directly related to a decrease in drug a ctivity. The most powerful application of multilayers is in the assessment of cytotoxicity. Solid tumour cell lines from different origins have been t reated with several conventional and investigational anticancer drugs. The data show that multilayers are more resistant to the drugs than the corresp onding monolayers, but there are substantial differences between the drugs depending on culture conditions, e.g, the difference was rather small for a drug such as cisplatin, miltefosine and EO9, a drug, which is activated un der hypoxic conditions. Gemcitabine was active against ovarian cancer but n ot against colon cancer, resembling the in vivo situation. This observation was not evident with monolayer experiments. Another interesting applicatio n is the possibility to perform drug combination studies. The combination o f gemcitabine and cisplatin proved to produce selective cell kill in H322 c ells (non-small cell lung cancer cell line). Neither of the drugs was indep endently able to produce similar effects. In summary, multilayer cultures a re relatively simple three-dimensional systems to study the effect of micro environmental conditions on anticancer drug activity. The model might serve as a base for a more rigorous secondary in vitro screening. (C) 2000 Elsev ier Science Ireland Ltd. AU rights reserved.