Spinal cord-derived neural stem cells cultured by serum-free suspension method：separation, cultivation and identification / 中国组织工程研究

ABSTRACT

BACKGROUND:Until now, there is yet no complete recovery from spinal cord injury in terms of structure and functional recoveries. Neurotrophic factors have limited effects on nerve regeneration. Currently, stem cel transplantation may be an effective way to repair spinal cord injury. OBJECTIVE:To separate, cultivate and purify mouse spinal cord-derived neural stem cels using serum-free suspension method folowed by morphological observation, immunofluorescence technology and multi-lineage differentiation experiments. METHODS:By using the suspension culture method, mouse spinal cord-derived neural stem cels at embryonic day 13.5 were cultured and purified. Cel morphology changes were observed under inverted microscope. Cel proliferation ability was detected using cel counting kit-8. Nestin and Sox2 expression was detected by immunofluorescence technology. Multilineage differentiation of spinal cord-derived neural stem cels at passage 4 was detected by natural differentiation method in order to prove the differentiation ability. RESULTS AND CONCLUSION: Serum-free medium suspension culture method was successfuly applied to separate spinal cord-derived neural stem cels. Cultured cels had good proliferative ability and highly expressed Nestin and Sox2 that was in accordance with the results of DAPI nucleus staining, suggesting the high purity of cels. After induction, the cels could express both Tuj1 and GFAP, indicating the cels had good differentiation potential. This experiment has successfuly established the isolation, culture, identification system of spinal cord-derived neural stem cels, providing experimental basis for subsequent studies of neural stem cels.