The Culture Of Neural Stem Cells In Rotating Wall Vessel Bioreactor

Neural stem cells (NSCs), as therapeutic agents, have important potential clinical applications for the treatment of central nervous system disorders such as Parkinson's, Huntington's. And they represent a potential source of non-transformed cells for drug testing. However, great quantities of NSCs are needed. And now the resource of NSCs is limited very much. Consequently there is an urgent demand to expand these cells in vitro. The aim of this study is to develop bioreactor protocols for the large-scale expansion of mammalian neural stem cells.In the study, NSCs were expanded in suspension in rotating wall vessel bioreactor (RWV) in large scale.Firstly, NSCs were obtained from the forebrain on embryonic day 13-15 (E13-15) KM mouse embryos and cultured in vitro. The results showed that these cells can grow in the way of neurospheres and expressed the characteristics of NSCs well. Then the study was ensured with precise and enough NSCs through the culture.Secondly, the pre-experiments about NSCs and RWV were performed. The optimal inoculation of 2 × 105cells/ml was obtained through the comparison of enlargement multiple of NSCs under different inoculations. Passaging time was determined in 5st-6st day (the neurosphere diameter of 100μm) after inoculation through the microscope observation and neurosphere diameter analysis. Meanwhile, the oxygen contents in the medium was analyzed by using the blood-gas analyzer and compared with the conditions in incubator. The results showed that the proper oxygen contents in RWV could be realized under the direct oxygen contact with the medium and the media circulated rate of 0.273ml/min. And the appropriate RWV rotating rate of 7-10rpm was determined through the analysis and experiments.Finally, based on the conditions above, NSCs were inoculated into RWV. NSCs cultured in RWV were evaluated in the aspects of growth state and ability of proliferation. And the property of stem cells was assayed with the methods of microscope observation, cck-8 kits, immunofluorescence and karyotype analysis. Meanwhile the metabolism of the cells was also examined. The results indicate that NSCs can successfully be expanded in suspension culture in RWV. NSCs in RWV grew well and remained their ability to differentiate into all three major cell types of the central nervous system (CNS)-neurons, astrocytes, and oligodendrocytes. Moreover, compared with NSCs cultured in T-Flask, NSCs cultured in RWV display moreenlargement multiple and less doubling time. In conclusion, rotating wall vessel bioreactor is an ideal three-dimensional system to culture Neural Stem Cells.