Macroporous hydrogels (MHs) hold great promise as scaffolds in tissue engineering and cell-based assails. In this study, the possibility of combination of three-dimensional (3D) cell culture with a miniaturized screening format was demonstrated oil human colon cancer HCT116, human acute myeloid leukemia KG-1 cells, and embryonic fibroblasts cultured on MHs (12.5 mm x 7.1 mm I.D.) in a 96-minicolumn plate format. MHs were prepared by cryogelation technique and functionalized by coating with type I collagen and by copolymerization with agmatine-based mimetic of cell adhesive peptide RGD (abRGDm). Cancer cells formed multicellular aggregates while fibroblasts formed adhesions on abRGDm-containing and collagen-MHs but not oil plain MHs, as was demonstrated by scanning electron microscopy. HCT116 and KG-1 cells grown as aggregates were more resistant to the treatment with cis-diaminedichloroplatinum (II) (cisplatin) and cytosine 1-beta-D-arabinofuranoside (AraC), respectively, during the first 18-24 h of incubation, than single cells grown oil unmodified AM HCT116 cells grown as 2D cultures in conventional 96-well tissue culture plates were 1.5- to 3.5-fold more sensitive to the treatment with 70 mu M cisplatin than cells in 3D cultures ill functionalized MHs. Further development of the described experimental system including matching of a specific cell type with appropriate extracellular matrix (ECM) components and 3D cocultures oil ECM-modified MHs may provide a realistic in vitro experimental model for high-throughput toxicity tests.