Research On Design, Fabrication, Detection And Applicationof Microfluidicchip Forneural Stem Cells

As the pluripotent stem cells, neural stem cells are undifferentiated and have self-renewal and replication in in the central nervous system. They are special cells group in the early development of the nervous system. Their findings bring the dawn for the further research on neural development and neural system diseases. In order to realize the various potential of NSCs, we should build the microenvironment of NSCs growth model similar to that in vivo. At present, the more conventional means are used in the research of NSCs, such as cultivation bottles or microtiter plates, Operation process is often tedious, time-consuming and reagent consumption is large, they are difficult to realize precise control. Microfluidic chips with its unique advantages can control cells under a few microns to a few hundred microns scales and could be employed to control proliferation and differentiation of cells in a microenvironment, which makes microfluidic chips one of the irreplaceable tools in the mechanism studies of stem cells. This paper designs and fabricates the microfluidic chips with the microsieves, explores the internal flow field and velocity distribution of chip under the microscale, researches the cultivation and differentiation methods of NSCs on the microfluidic chip, establishes the microenvironment that suits for NSCs. The main research results are asfollows:1. Use soft lithography to fabricate PDMS chip. Modify SU-8 glue, spin rate: 500 rpm 18s, 1500 rpm 30s. Prebake:65℃10min, 95℃1h. Exposure:310s. Postbake:65℃ 5min, 95 ℃ 20 min, develop for 3min. Use injection moulding, put the PDMS elastomer on template, get chip after packaging and bonding.2. Make the fluid mechanics analysis on the microsieves through the Micro-PIV technology. We inject fluorescent particles in microchannel,analyze flow field and the velocity vector distribution. Speed statistics and flow field distribution are similar to Ansys finite element simulation. Research results show that the flow field distribution of microsievesis stable and without mutation. The velocity of internal microsieves decreases, it effectively decreases the damage of shear force produced by liquid flowing to cells and microsieves are beneficial to maintain cell inoculation density, cells can be distributedevenly in the microchannel.3. Use the double package method of PDL and Laminin on microfluidic chip and culture NSCs on the chip. After continuous culture, cells grow in reunion, survival rate is high and the neurites are raised. The microsievespromote uniform spatial seeding of small clusters of cells,the average density is 100/sieve. Due to the effect of blocking and capture of microsieves, cell cultivation density becomes slightly higher than that in the microchannel.4. We culture the NSCs and GABA neural stem cell RMNE6 on the microfluidic chip and induce differentiation, used PDL and Laminin packaging the chip. Through this method, cells have a good adhesion growth, the average adhesion rate reach 85.7%. After continuous culture, the neurites are raised and connect each other. Cells grow together, cell density in the microsieves is larger than that in the mcrochannels. Some part, we can observe neuron and neurofilament.