Collagen protein is an ideal scaffold material for the transplantation of neural stem cells.In this study,rat neural stem cells were seeded into a three-dimensional collagen gel scaffold,with suspension cultured neural stem cells being used as a control group.Neural stem cells,which were cultured in medium containing epidermal growth factor and basic fibroblast growth factor,actively expanded and formed neurospheres in both culture groups.In serum-free medium conditions,the processes extended from neurospheres in the collagen gel group were much longer than those in the suspension culture group.Immunofluorescence staining showed that neurospheres cultured in collagen gels were stained positive for nestin and differentiated cells were stained positive for the neuronal marker βIII-tubulin,the astrocytic marker glial fibrillary acidic protein and the oligodendrocytic marker 2’,3’-cyclic nucleotide 3’-phosphodiesterase.Compared with neurospheres cultured in suspension,the differentiation potential of neural stem cells cultured in collagen gels increased,with the formation of neurons at an early stage.Our results show that the three-dimensional collagen gel culture system is superior to suspension culture in the proliferation,differentiation and process outgrowth of neural stem cells. Collagen protein is an ideal scaffold material for the transplantation of neural stem cells. In this study, rat neural stem cells were seeded into a three-dimensional collagen gel scaffold, with suspension cultured neural stem cells being used as a control group. Neural stem cells , which were cultured in medium containing epidermal growth factor and basic fibroblast growth factor, actively expanded and formed neurospheres in both culture groups. In serum-free medium conditions, the processes extended from neurospheres in the collagen gel group were much longer than those in the suspension culture group. Immunofluorescence staining showed that neurospheres cultured in collagen gels were stained positive for nestin and differentiated cells were stained positive for the neuronal marker βIII-tubulin, the astrocytic marker glial fibrillary acidic protein and the oligodendrocytic markers 2 ’, 3’-cyclic nucleotide 3’-phosphodiesterase. Compared with neurospheres cultured in suspension, the differentiation potential of neural stem cells cultured in collagen gels increased, with the formation of neurons at an early stage. Here results show that the three-dimensional collagen gel culture system is superior to suspension culture in the proliferation, differentiation and process outgrowth of neural stem cells .