| Neural stem cells (NSCs) have great potential in the clinical treatment of neuro degenerative diseases, nerve damage and other diseases. However, currently, the sources of NSCs are limited, so it is difficult to apply them in a large-scale due to the insufficient cell number. Traditional in vitro expansion methods including neurospheres suspension culture and adherent culture, have disadvantages, such as, expanding rate is not quite high, can not well simulate the real circumstance of living organism and so on. Therefore, their limitations are becoming more and more notable.Calcium alginate micro-gel bead as a carrier of immobilized cell and a technology have many advantages, such as, low cost, simple operation, mild conditions for immobilization, small diffusion resistance of nutritional substance, metabolite, biocompatibility and many other advantages. At present, the calcium/alginate microbead as a technique has gained common attention and wide application in vitro expansion and in vivo transplantation, but it is seldom reported that it is appied to encapsulate NSCs as a kind of cell carrier.Gelatin is a kind of natural polymer material, its structure is similar to that of the extracellular matrix (ECM) and it has good biocompatibility. Therefore, it has a wide range of applications in the biomedical field. In this study, the alginate/gelatin microbeads were prepared with sodium alginate and gelatin as the matrix material, in order to obtain a better culture system which is more conducive to cell growth and proliferation compared with the current cell carrier.In this study, in order to explore the feasibility of this composite microbeads used in NSC culture and expansion in vitro, first, the effect on mechanical strength of the microbeads caused by the concentration of sodium alginate, gelatin and CaCl2 and other preparation parameters was studied by using a self-made puncture force tester. The soaked strength properties of microbeads resistant to medium were also researched. The permeance property was researched by building a diffusion model and studying the diffusion of glucose and bull serum albumin (BSA) in the microbeads. An equation related the diffusion coefficient and concentration of sodium alginate, gelatin and CaCl2 was calculated. The optimized parameters for preparing microbeads with a proper diffusivity in static culture were determined by a comprehensive analysis of parameters, such as strength and diffusion performance. And the parameters are 1.5%(wt%) sodium alginate,0.5% gelatin,4%(wt%) CaCl2, respectively.Next, the encapsulated NSCs were cultured by adopting optimized preparation parameters, the cell morphology and the nestin expression were both compared before and after encapsulation culture. The results indicated that there was a good biocompatibility between alginate/gelatin microbeads and the NSCs, the cells harvested after 11 days were nestin-positive, and can differentiate into neurons, astrocytes and oligodendrocytes, suggesting that cells still remain the features of neural stem cells. Meanwhile, the multiplication of different encapsulating density was compared and the optimal culture density was 1.5x105 cell·mL-1. Moreover, the oxygen consumption rate of encapsulated NSCs, and the parameters including glucose consumption and the alteration of suface antigen, were all assayed systematically, which explored the technological parameters and provided a theoretical basis for the application of microbead technology in the field of large-scale dynamic NSCs culture in vitro.
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