Culture Of Neural Stem Cells In Collagen Gel And Collagen-Chitosan Scaffolds

There are two traditional cultrue models for neural stem eells(NSCs),suspension and monolayer culture.However,the unevadable defect of the models restrict their application. Recently,the three-dimensional culture model is being the hotspot in the field of cell culture. Cells can grow in a three-dimensional network and form a structure that similar to the body.Three-dimensional culture model can not only induce the cells to differentiate directionally and maintain the phenotype after differentiation,but also be expected to build cell three-dimensional analogues and equivalents,corresponding with all kinds of tissues and organs in vitro.So,this culture technology is very significant for tissue engineering and clinical application.In the paper,the three-dimentioal culture model was applieated to culture NSCs.The nature biomaterial collagen was used to compound with chitosan in order to make the scaffolds.The biocompatibility of the collagen was evaluated firstly,including the security and functionality.Then,collagen and ehitosan was fully mixed,freeze-dried,cross -linked and freeze-dried again to form the scaffolds.The microstructure of the scaffolds was observed by scanning electron microscopy.The biocompatibility of NSCs with composite scaffolds was proved by the cell experiments,and the immunofluorescence test was performed by laser scanning confocal microscopy(LSCM).The main results are as follows:(1)The biocompatibility of collagen and NSCs:Results indicated that the single NSCs dispersed in collagen gel evenly observed via inverted phase contrast microscope during the initial culture,and obvious cluster masses appeared 3 days later.The cell viability in collagen gel was higher than that of traditional culturing methods,and most cells showed nestin-positive and BrdU-positive.The appropriate concentration of collagen is 0.5mg/ml and the suited cell density is 1×10~6cells/ml.(2)The preparation of the collagen-chitosan scaffolds and their features:the freeze-drying technology was adopted to prepare the eollagen-chitosan scaffolds.The suitable volume ratio between collagen and chitosan is 7:3 for cell growth after the comprehensive evaluation of the scaffolds' average pore sizes,porosity and degradation rate.(3)Statistical analysis of datas showed that there was a better compatibility between cells and the scaffolds with volume ratio 7:3 since NSCs can adhere,expend and grow well in the scaffolds.The cell viability was 73±2.8%after NSCs were cultured in scaffolds for 6 days. The fluorescence staining results showed that NSCs cultured in the scaffolds can differentiate into neurons and gliocyte,and maintain the characters of stem cell. From the above experimental results,we can infer that the collagen - chitosan composite scaffold can be applied in the nerve tissue transplantation in clinical application.