Culture and amplification of neural stem cells of mice with serum medium containing basic fibroblast growth factor / 中国组织工程研究

ABSTRACT

BACKGROUND: Neural stem cells are a kind of special cells possessing self-renewal and multi-directional differentiation potential. They are ideal carriers for studying the occurence, development and development rule of nervous system and their inherent regulation mechanism of molecular biol ogy and cytology. They have wide applicative prospect in the treatment of nervous system disease and injury. Investigating a set of convenient, effective and useful culture system of neural stem cells cultured in vitro is the prerequisite and basis for application. OBJECTIVE: To observe the effect of serum culture medium containing basic fibroblast growth factor on the in vitro culture of neural stem cells of mice. DESIGN: Single-sample observation. SETTING: Department of Histology and Embryology, Youjiang Nationality Medical College. MATERIALS Five 2-month-old Kunming white mice of either sex were used in this experiment. METHODS: This experiment was carried out at the Department of Histology and Embryology, Guangxi Medical University from June 2003 to May 2005. ① The cerebral cortical cells were isolated from cerebral cortex and cultured in vitro of the adult mouse cerebral cortex and made into cell suspension , then put in the DMEM/F12 (11)culture medium (containing fetal bovine serum of 0.15 volume fraction, basic fibroblast growth factor of 20 μg/L, penicillin of 100 u/mL and streptomycin of 100 u/mL ) .The attachment culture method was used to acquire the clone cells. ② Expression of nestin antigen of clone cells was detected with immunohistochemical technique. MAIN OUTCOME MEASURES: ① Morphological observation of cultured cells. ② Observation of cultured cells stained by haematoxylin and eosin . ③ Identification of neural stem cells. RESULTS: ① Morphological observation of cultured cells After inoculation, most of the cells attached to the wall within 12 hours, cells were thin and flat. 3 days after inoculation, cells began to grow and proliferate. There were several scores to several hundreds of cell clones on the 5th day after inoculation and cells covered 80%-90% of the bottom of the bottle on the 7th to 8th days. After passage, the cells still grew adhesively with regular cellular morphology and clear borderline. ② Observation of cultured cells stained by haematoxylin and eosin It was shown that the cells presented round or ellipse. There was little cytoplasm, presenting acidophily. Nucleus was big and round, single in the center, deep stained.③ Identification of neural stem cells Immunofluorescent detection showed that cell nestin antigen positive. CONCLUSION: After cultured in serum culture medium containing basic fibroblast growth factor, cerebral cortex cells possess very strong reproductive activity and express nestin antigen, still possessing the characteristics of neural stem cells.