Three-dimensional Culture Of Neural Stem Cells And Construction Of Neural-like Tissues

Two traditional models for neural stem cells (NSCs) culture, suspension and monolayer, are commonly used. However, the two models are difficult to mimic the in vivo three-dimensional microenvironment. Nevertheless, three-dimensional cell culture system can provide much a microenvironment for cells to secret extracellular matrix and grow better, so that the closely linked cells can form organization-like construct. Therefore, we applied three-dimensional culture model to culture NSCs and constructed neural tissue-like structures.The NSCs from rat hippocampus as seeding cells, and collagen hydrogel as scaffods were used in the experiment. Firstly, the growth and metabolism of cells were compared in the three-dimensional collagen culture system, traditional monolayer culture system and suspension culture system. Then, the proliferation and differentiation culture media were applied respectively during the intial and later culture period to improve the survival time of the collagen-entrapped cells and to form tissue-like neural network under static conditions in vitro. The proliferation of cells cultured in the tissue-like neural network was detected by cck-8 kit (Cell counting kit-8, cck-8). And the immunofluorescence test and cell live/dead stain were performed by laser scanning confocal microscopy and viability-cytotoxicity kit to detect the viability and differentiation of cells in the constructs. Finally, a preliminary study was carried out for NSCs three-dimensional perfusion culture.The results showed that the NSCs fold expansion cultured in collagen gel was the highest, which was about 70 times after 3 days, while those for the suspension and monolayer culture were 2.5 times and 35 times respectively. The results of glucose and lactate metabolism showed that the specific productive rates of glucose and lactic acid were the lowest for NSCs cultured in collagen gel. Furthermore, the live cell ratio maintained around 82%in neural network pattern after 42 days culture. And the cells uniformly distributed in gels and differentiated into neurons, astrocytes and oligodendrocytes. Additionallly, the cells adhesion ratio was significantly improved in LN-pretreated collagen sponge scaffolds compared with culture medium pretreated scaffolds. Finally, NSCs cultured in perfusion bioreactor grew in cell-cluster pattern, distributed uniformly and expressed Nestin positively. Perfusion bioreactor was benefit for nutrient supply and pH sustaining so that NSCs can be cultured in a mimic physical environment. It can be concluded that the three-dimensional collagen culture system is benefit for the proliferation of NSCs and their metabolism. The three-dimensional neural network-like constructs were similar to the neural tissues in morphology and cell composition. LN-pretreated collagen sponges can promote NSCs adhesion. And perfusion bioreactor was benefit for cells distribution, proliferation and maintaining the characteristics of NSCs. They thus have a potential to be used for drug screening, detection of environment toxins and replacement therapy.