Neural Stem Cells In Vitro System

Objective:To explore efficient technology and conditions of neural stem cells(NSCs) culture by replacing cytokines, and set up the cultured system in vitro for neural stem cells, providing a base for establishing cell resource library in practice.Methods:1. Unicell suspension was made by isolating tissue from hippocampus. Cells from hippocampus were cultured in serum-free DMEM/F12 medium with 20ng/ml bFGF, 20ng/ml EGF and 2%B27.Cells in primary culture and in passage were detected Nestin antigen by immunocytochemistry methods. Andβ-Tublin and GFAP antigen were also detected respectively by similar ways after the cells differentiated. Cells labeled by Brdu were transplantated into homogenous mice's brain for detecting their surviving.2. The embryo fibroblast cells, Sertoli cells and bone marrow stromal cells were isolated and cultured, and were made into feeder layers by 10μg/ml of mitomycin C.3. There were five groups in this experiment, and the growth curves were based on the results above.Results:1. Numerous neurospheres formed and developmented in cytokine- cultured system, floating in the medum. New neurospheres could also form after high passage, and all cultured cells showed nestin- immunopsotive. Some differentiated cells showedβ-Tublin, but others GFAP-positive cells were also observed. Immunocytochemistry staining showed that the cultured cells appeared Brdu-immunopsotive after Brdu-labeling, whether in primary culture or after passage. When these cells were transplanted into the mouse brain, many Brdu-positive cells were observed easily at hippocampus and lateral ventricle after 7days.2. Successful feeder layers were building by embryo fibroblast cells, Sertoli cells, and bone marrow stromal cells.3. Neural stem cells can proliferate normally in all these three kind of feeder layers, but the effect in Sertoli cell feeder layer wasn't better than that in others. Proliferating action of neural stem cells in cytokine group was stronger than that in feeder layer groups. The cells' differentiating was obvious in control group.Conclusion:1. Neural stem cells can be isolated from newborn mouse's hippocampus.2. The transplanted NSCs can survive in brain.3. Neural stem cells proliferated stably and maintained undifferentiated characteristic in the three feeder layers. The effects of embryonic fibroblasts and bone marrow stromal cells feeder layers were better than that of sertoli cell feeder layers.4. Although cytokine group is superior to another group, it is expensive. And the feeder layer group is superior to simulate the micro-environment in vivo by producing many kinds of cytokines. It is worthy of being spread in ordinary laboratories.