A Multilayer Vertical Perfusion-based Cell Culture Device For Drug Screening

Cell-based assay has become the main method of drug screening, because of higher accuracy and less interference factors. Moreover, it is able to provide good mimics of the in vivo environments. Microfluidics (or lab-on-a-chip), as a flexible combination and a scale of integration of a variety of units, has been adopted as an accepted and applied form of cell-based drug screening. Compared to the conventional technology, microfluidics has potential to eliminate the preparation and distribution of drug solutions of different concentrations, and greatly simplify the process of cell loading, cell stimulated, washing and cell labeling, to significantly reduce cell and reagent consumption. The main research contents in this dissertation are as follows:We present and design an integrated multilayer cell culture device based on vertical perfusion cell culture, which is integrated with a concentration gradient generator based on serial dilution, a vertical perfusion-based cell culture microchamber, and pneumatic microvalves. In this section, the main research contents include:analysis and simulation in theory of concentration gradient generator, structural design of cell culture microchamber, simulation of the fluid shear stress, simulation of cells distribution and form of cells stopped by microcolumns, research on the deformation of the membrane, and design of the air control system.The SU-8 thick-photoresist master molds and silicone mold under dry etching were fabricated successfully with high quality of patterning. After treated, the molds were hydrophobic and the PDMS chips made by replica molding were much easier to be peeled off from the molds. The fabrication parameters of microvalve membrane the thickness of which is below 100μm have been analyzed. The mechanism for alignment of multilayer microfluidic device has been proposed to achieve quick and easy alignment. Finally, the multilayer microfluidic device with high bonding strength has been obtained.In the experiment section, the performance of CGG in accordance with the fluorescence intensity has been verified. The real concentration gradient generated in each outlet showed a relatively high linearity (R was 0.9959), and the relative error to the designed concentration was less than 10%. The A549/DDP cells were cultured successfully in the microchambers of the multilayer PDMS device. After the cells have been induced by Cisplatin varying in concentration, the viability rate of cells was 92.3% at a lower drug concentration (9.0μM), while that was 61.2% at a higher drug concentration (91.9μM). The results showed that this integrated microfluidic device enabled to provide a well-defined and stable culture environment, and that Cisplatin was able to induce A549/DDP cell apoptosis and induced effect was in a concentration-dependent mode.