Detection of imatinib and doxorubicin resistance in k562 leukemia cells by 3d-electrode contactless dielectrophoresis

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2013
Demircan, Yağmur
Dielectrophoresis (DEP) is a MEMS-enabled technique defined as the relative movement of particles and medium under nonuniform electric field. DEP can be utilized for biomedical applications manipulating biological particles based on their dielectric properties and sizes. For example, cancer cells are different than normal tissue cells in terms of both their sizes and dielectric properties, leading to the potential use of DEP in early cancer detection. DEP can provide even the separation of cells with similar size, based only on the differences dielectric properties such as, multidrug resistant (MDR) cancer cells. MDR is a condition enabling a cancer cell to resist distinct drugs or chemicals of a wide variety of structure or function targeted at eradicating the cell. In this case, patients do not respond the chemotherapy. Therefore, MDR detection in early stages is crucial to choose the most proper treatment and to accelerate the recovery period. This thesis presents the detection of imatinib and doxorubicin resistance in K562 leukemia cells by 3D-electrode contactless dielectrophoresis (DEP). The main objective of the thesis is to detect MDR in cancer cells based on their dielectric properties with continuous flow DEP in label free manner. For the proof of cell manipulation by DEP based only on dielectric properties, the 1st generation devices’ design was achieved with the iterations in finite element (FEM) simulations in COMSOL. In this design, 3D-electrodes were used in the form of reciprocal short and long electrodes to provide nonuniform electric field in channel length and width. Fabrication flow for this design was developed and fabrication of these devices was performed. Very thin (~0.3 μm) parylene layer was coated on electrodes to prevent Joule heating and cell damaging. Testing of the 1st generation devices was carried out with the viable and nonviable yeast cells. Therefore, triple shell cell modeling of them was carried out through MATLAB before testing. It was reported that viable yeast cells were trapped with a purity of 96.8 % at the crossover frequency of nonviable yeast cells (1.45 MHz), at which nonviable yeast cells carried away with the capillary flow inside microchannel. Considering the problems associated with the fabrication and experimental setup of the 1st generation devices, a 2nd generation DEP device was designed for the detection of MDR in K562 cells. In the 2nd generation design, 5 V-shaped parylene obstacles were utilized to direct the cells into DEP area hydrodynamically eliminating the usage of the external pressure actuation devices, utilized in the 1st generation. Moreover, 3D separated electrodes, which provide vi uniform DEP force through the channel depth, were utilized in side walls along the channel length to increase the efficiency of DEP device. A thin parylene coating (~0.3 μm) on the electrodes provided the insulation of electrodes as in the 1st generation devices. Due to very thin parylene coating, the necessary voltage (minimum 5 Vpp) for DEP operation is considerably lower than the voltage of other contactless DEP devices reported in the literature. Before testing of this device, dielectric modeling of imatinib and doxorubicin resistant, and sensitive K562 cells were achieved by double shell cell modeling through MATLAB. While the trapping of doxorubicin and imatinib resistant K562 cells was observed at the crossover frequency (48.64 MHz) of sensitive K562 cells, sensitive K562 cells were not trapped under the same experimental conditions. This result shows that the separation of imatinib and doxorubicin resistant K562 cells can be achieved with this design. Moreover, performance tests of the 2nd generation devices are made with changing the key parameters of design: the flow rate, voltage magnitude and cell concentration. Device has a trapping ability at minimum 5 Vpp voltage and 20 μl/ min flow rate when cell concentration is 625000 cells/ml. Finally, the effect of drug (doxorubicin) resistance level on the DEP response of K562 cells was examined. The trapping characteristics of cells which have different degree of drug resistance were differentiable according to the preliminary test results. It is worthy to note that all test results are consistent with theoretical expectations and numerical solutions in finite element simulations. In conclusion, the proof of DEP concept based only on dielectric properties was achieved. Two different DEP device design were utilized to improve the quality of fabrication and test results. The detection of imatinib and doxorubicin resistance in K562 cells were achieved with different performance parameters, including voltage magnitude, flow rate and cell concentration. The degree of drug resistance in cancer cells was examined for the first time in the literature in electrical manner. With the further improvements in design, the detection of MDR in real patient blood is the ultimate goal.

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Citation Formats
Y. Demircan, “Detection of imatinib and doxorubicin resistance in k562 leukemia cells by 3d-electrode contactless dielectrophoresis,” M.S. - Master of Science, Middle East Technical University, 2013.