Study Of Cell Differentiation And Cellular Interaction On Microfluidic Device

Due to its advantages of low sample consumption, rapid analysis, high throughput, especially the ability to manipulate small size samples, microfluidic technology shows great potential in the field of cell research. Based on microfluidics combined with fluorescence microscopy imaging and mass spectrometry, this thesis carried out researches on cell differentiation and cell-cell interaction.Firstly, the research progress in the application of microfluidic technology and microchip-mass spectrometry(chip-MS) in the field of cell analysis was reviewed. The microfluidic technology has a great potential applied in the study of cell differentiation and cellular interaction.Next, the standard soft lithographic and replica molding techniques, curing agent dispersion method and polydimethylsiloxane(PDMS) glue method was adopted to fabricate the polycarbonate-membrane-integrated microfluidic device. We optimized the producing conditions for fabricating the membrane-integrated device, and investigated the mass transfer ability across the membrane and the integration ability with other chips. The polycarbonate membrane integrated in the microfluidic device allowed almost all biochemical factors to diffuse across it, which is not only essential in cell culture but also in mimicking physiological conditions as cells constantly receive signals from soluble environments. Besides, mES cells were cultured on polycarbonate membrane in top channels while the bottom channels were connected with syringes driven by a pump, realizing mouse embryonic stem(mES) cells continuous flow culture and induced differentiation under low shear force condition on chip.Finally, a polycarbonate-membrane-integrated microfluidic device with cellular metabolite detected by high sensitivity mass spectrometer for cell co-culture and cellular metabolic analysis was developed. This established platform provided us with a chemical insight into understanding cellular interaction. The physiological process, adrenal gland secreting epinephrine under the regulation of sympathetic nerve, was simulated on the membrane-integrated microfluidic chip with differentiated PC12 cells acting as the sympathetic nerve and 293 cells acting as the adrenal gland. We optimized the differentiation condition for PC12 cells, realized co-culture of differentiated PC12 cells and 293 cells on the membrane-integrated microfluidic device, and successfully detected the secreted cellular metabolite molecule epinephrine by Nano-ESI-MS. Specially compared to similar works, we did not need sample pretreatment prior to MS detection, which reduced sample consumption and analysis time. This membrane-integrated device could be used in the study of both known and unknown essential signaling factors in important regulation pathways for disease monitoring and drug development.