Zhiying Zhao1, Haitao Hu, Shehui Shi. 1. Department of Anatomy, Medical College of Xi'an Jiaotong University, Xi'an Shaanxi, 710061, P. R. China.
Abstract
OBJECTIVE: To establish a better method of isolating and culturing of neural stem cells (NSCs) in neonatal rat brain. METHODS: Tissue of brain was isolated from neonatal rats. Different medium and culture concentration were used to culture NSCs of neonatal rat. The culture concentration used were 1 x 10(4), 1 x 10(5), 1 x 10(6), and 1 x 10(7)/ml respectively. Ingredient of medium was classified into group 1 to 8 respectively according to whether to add 2% B27, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) as well as the difference in culture concentration. The cells were induced to differentiate as to be confirmed as NSCs, and then were checked by phase contrast microscopy and identified by immunocytochemistry. RESULTS: The cells isolated and cultured gathered into neurospheres. The cells were capable of proliferating and maintaining long-term survival in vitro. The cells could be differentiated into neurons and glia. It was to the benefit of the survival of NSCs to add 5% fetal bovine serum(FBS) into the medium at the beginning of the culturing. When 10% FBS was added into the medium, the neurospheres differentiated quickly. When concentration 1 x 10(6)/ml was used, the growth rate of the cells was the highest of all the concentrations. Reasonably higher cell concentration promoted the proliferation of NSCs. It was necessary to add 2% B27, EGF, and bFGF into the medium. The cells had the best growth when 2% B27, 20 ng/ml bFGF and 20 ng/ml EGF were added into the culture medium. EGF and bFGF had cooperative effect. CONCLUSION: A better method of isolating and culturing of NSCs in neonatal rat brain is established and the foundation for future research is laid.
OBJECTIVE: To establish a better method of isolating and culturing of neural stem cells (NSCs) in neonatal rat brain. METHODS: Tissue of brain was isolated from neonatal rats. Different medium and culture concentration were used to culture NSCs of neonatal rat. The culture concentration used were 1 x 10(4), 1 x 10(5), 1 x 10(6), and 1 x 10(7)/ml respectively. Ingredient of medium was classified into group 1 to 8 respectively according to whether to add 2% B27, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) as well as the difference in culture concentration. The cells were induced to differentiate as to be confirmed as NSCs, and then were checked by phase contrast microscopy and identified by immunocytochemistry. RESULTS: The cells isolated and cultured gathered into neurospheres. The cells were capable of proliferating and maintaining long-term survival in vitro. The cells could be differentiated into neurons and glia. It was to the benefit of the survival of NSCs to add 5% fetal bovine serum(FBS) into the medium at the beginning of the culturing. When 10% FBS was added into the medium, the neurospheres differentiated quickly. When concentration 1 x 10(6)/ml was used, the growth rate of the cells was the highest of all the concentrations. Reasonably higher cell concentration promoted the proliferation of NSCs. It was necessary to add 2% B27, EGF, and bFGF into the medium. The cells had the best growth when 2% B27, 20 ng/ml bFGF and 20 ng/ml EGF were added into the culture medium. EGF and bFGF had cooperative effect. CONCLUSION: A better method of isolating and culturing of NSCs in neonatal rat brain is established and the foundation for future research is laid.