| Neural stem cells (NSCs) are self-renewable and have the potential to differentiate in multi-directions. The discovery of NSCs has brought great hope for further research on the development of the nervous system, the treatment of neurodegenerative diseases, and clinical transplantations. However, the mechanisms of NSCs proliferation and differentiation are still not well understood. Constructing NSCs model in vitro can be a valuable tool for exploring these mechanisms, which may contribute a lot to the fields of neuroscience and neural tissue engineering.Traditional in-vitro NSCs culture methods include monolayer culture and the "neurosphere" suspension culture. However, these methods fail to embody the biological characteristics and functions of NSCs in vivo. Growing cells in three-dimensional (3D) matrix is considered a more meaningful method that better simulates the microenvironment of mammalian brain. Constructing3D NSCs model in vitro would provide great value to theoretical and practical applications for promoting the studies of mammalian nervous system development, clinical transplantation of nervous tissues, and drug screening. Currently, the research on3D NSCs culture is still at the stage of experimental research, and there are no standard experimental methods. This paper mainly focuses on three-dimension NSCs culture in microfluidic chip systems, and relevant proliferation and differentiation control issues. For this purpose, the following work has been carried out.Mouse NSCs (mNSCs) were isolated from the hippocampus of embryonic day-14Kunming mice fetal brain by the method of primary culture. Then, the growth trend and the distribution of neurosphere sizes of five mNSCs suspension cultures with different cell density were investigated. The results showed that the best inoculation density of the primary mNSCs was1×105cells/mL. Subcultured cells could stably express NSCs characterized protein (Nestin and Sox2) and were able to differentiate into neurons (β-tubulin-positive), astrocytes (GFAP-positive). and oligodendrocytes (01-positive) induced by10%fetal calf serum (FCS) medium.After careful screening, one serum-free medium (MA) was selected for mNSCs amplification in vitro. In order to obtain enough mNSCs for constructing the three-dimension NSCs model, orthogonal tests were used to screen and determine the optimum concentration and ratio of the added components in culture medium MA that can promote mNSCs amplification. The tests displayed that the maximum cell amplification result of the mNSCs cultured in MA can be as high as (7.25±0.23)×105cells/mL (p<0.01).7.25times of the initial inoculum density ((1.0±0.12)×105cells/mL). Besides, immunofluorescence staining showed that the Nestin positive ratio of the mNSCs cultured in the MA was94.2±3.5%. which indicated that the mNSCs cultured in MA can maintain the stem cell characteristics. Also, the glucose/glutamine ratio in MA was proved to be perfect for the growth and metabolism of mNSCs. Which reconfirmed MA to be an excellent medium which was appropriate for mNSCs amplification in vitro.A two-step method for constructing3D mNSCs model under static conditions in vitro is proposed. The two-steps culture method was used to simulate the physiological environment of different phases of neurogenesis in vivo. The3D scaffold was0.5ing/ml, type1collagen hydrogel based. Firstly, the medium consisting of DMEM/F12/RPMI1640(1:1:1) supplemented with EGF/bFGF/BSA/Lipids was used to expand the mNSCs embedded in collagen in gel in96-well plates until the average diameter of cell clusters reached50-100μm. Secondly, the initial medium was replaced by medium MC. which is NB/B-27supplemented with bFGF and BDNF. The results showed that the cells in collagen presented neural-like morphology and maintained live cell rate around82%. The cell-collagen constructs were examined by immunofluorescence and immunohistochemistry tests, which showed14.17±2.6%cells still maintained the character of mNSCs.40.93±1.8%cells differentiated into neurons, and27.13±1.6%cells differentiated into astrocytes. Our3D neural-like tissue constructs were similar to the neural tissues in morphology and cell compositions.Finally, the lnicrofluidic cell culture system was used for the three-dimension mNSCs culture. A microfluidie chip with micro-fabricated pillar arrays was designed for the in-situ formation and immobilization of3D mNSCs-collagen construction. The cell-collagen constructs in mierolluidic chip were detected by immunofluorescence test after7days of culture. And. the concentration changes ol lactic acid and glutamine in the3D culture systems were also monitored. The results showed that part of the eells in the center of the3D constructs expressed Nestin protein, and others differentiated into neurons and aslrocytes. which indicated that mNSCs can be self-renewable and differentiate under engineered3D microfluidics culture system. The concentration of lactic acid was steadily controlled below1.0mmol/ml and the concentration ofglutamine was remained around2.0mmol/tnL. which proves that the mierolluidic cell culture system can provide a stable microenvironment for3D mNSCs. In contrast, under the static culture condition, the concentration of lactic acid fluctuated between2.5～4.5rnmol/mL with batch and medium exchange modes, and the concentration of glutamine decreased with cells consumption and needed to be supplemented constantly. The two-step method is easy, stable and fast in building3D mNSCs models on microfluidic chip. And. the3D cell constructs in the chip can be observed clearly under inverted microscope. More importantly, the3D mNSCs model proposed here is similar to the neural tissue in morphology and cell compositions. Therefore, it has great potential for safety pharmacology, drug discovery and toxicity testing. |
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