Dynamic single-cell analysis using microfluidic biochips pertaining to drug discovery

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(Thesis) Ph.D.
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New microfluidic methods have been developed for the dynamic single-cell analysis pertaining to drug discovery that includes drug efficacy, drug safety and drug transport. A microfluidic approach that integrates various single-cell operations as well as the cytosolic calcium measurement on one microchip has first been developed. The on-chip dye loading was performed to minimize cell damage. The cytosolic Ca2+ concentration ([Ca2+]i) of a single cell was quantified for the first time in a microfluidic chip. To study drug efficacy, the microfluidic [Ca2+]i measurement was applied for the real-time detection of cytotoxicity of drug candidates on single leukemia cancer cells (RAW cells). The microfluidic results have been confirmed by the conventional cytotoxicity assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). Isoliquiritigenin (IQ, an herbal ingredient of licorice) has been found to have an antiproliferative effect on leukemia cells. The microfluidic single-cell analysis not only reduces reagent cost and the number of cells needed, but also reveals some phenomena due to cell heterogeneity that cannot be observed in bulk analysis. As for drug biosafety, the microfluidic [Ca2+]i measurement was employed to study the cardiotoxicity of drug candidates on single cardiac myocytes. A new microchip containing a weir structure to improve the cell retention of cardiac myocytes was fabricated by a new low-cost method, with the weir structure dimensions estimated by a mathematical model. It has been found that IQ could have less cardiotoxicity as an anticancer drug candidate. To study drug transport, a microfluidic same-single-cell analysis (SASCA) method has been developed to investigate multidrug resistance (MDR) modulation in single leukemia cells (CEM cells). I put forward the new concept of SASCA to address the issue of cell heterogeneity, and successfully demonstrated its advantages in the MDR drug efflux study. It has been found that the herbal ingredient artesunate, but not IQ, has a MDR reversal effect. The microfluidic results have been confirmed by flow cytometry. All these microfluidic approaches were developed with an ultimate goal to improve cancer chemotherapy.
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