Stepwise microfluidic channel chips for cell-matrix adhesion characterization in multiple shear stress zones

Abstract

This thesis presents two types of stepwise microchannel chips for cell-matrix adhesion characterization in multiple shear stress zones: cell adhesion chip based on visual detach-ment observation and cell adhesion chip based on visual detachment observation and impedance analysis. The previous cell adhesion chips based on visual detachment observation cannot analyze the response of the cells simultaneously in multiple shear stress zones or needs complex structure for formation of the multiple shear stress zones. In addition, since these previous cell adhesion chips are dependent on visual images, they cannot analyze the cell-matrix adhesion state like as contact area and gap between cells and matrix. Therefore, we propose two types of the cell adhesion chips using simple stepwise microchannel with multiple shear stress zones: cell adhesion chip capable of analyzing the cell detachment rate based on visual detachment observation and cell adhesion chip able to characterize both cell detachment rate and cell-matrix adhesion state based on visual detachment observation and impedance analysis. The cell adhesion chip based on visual detachment observation has stepwise widths between an inlet and outlet port. The chip is able to generate the multiple shear stress levels by varying width of the microchannel. After adhering cells stably on the microchannel, we apply the shear stress levels of 35, 41, 47, 54, 60 $dyne/cm^2$ to the cells by flow of culture media. And then, we characterize the cell detachment rate using visual detachment observa-tion. In the experimental study, we used A549con and A549E-cad (E-cadherin knock downed A549 con cells) human lung cancer cell lines. The A549E-cad cells showed about three to five times lower detachment rate than A549con cells at the shear stress levels of 35~60 $dyne/cm^2$ , thus implying E-cadherin affected in cell-collagen adhesion and strengthened the bond. The cell adhesion chip based on visual detachment observation and impedance analysis consists of stepwise microchannel and electrode-patterned glass substrate. And the external electrode is plugged in inlet port of the microchannel and it is used to analyze the impedance response with patterned electrode. After attaching the cells on each electrode allocated in stepwise microchannel, we treat the cells with 5 % ethanol media in static condition (0 $dyne/cm^2$) and dynamic condition (1.2, 1.8, 2.5, 3.1, 3.8 $dyne/cm^2$). And then, we characterize the cell detachment rate and cell-matrix adhesion state using visual detachment observation and impedance analysis. In order to verify the toxic effect, we finally measure the cell death rate by staining the cells with Propidium Iodide (PI). In the experiments, we used MDA-MB-231 human breast cancer cells. The cell detachment rate showed steep decrease (about 5.0 %) at the shear stress level of 1.8 $dyne/cm^2$. In the impedance analysis, we estimated normalized $R_{cells}$ and normalized $C_{cells}$, electrical parameters related to cell-matrix adhesion state. Normalized $R_{cells}$ and normalized $C_{cells}$ decreased rapidly as about 8.4 % and 41.2 %, respectively, at the shear stress level of 2.5 $dyne/cm^2$, which means that the change of cell-matrix adhesion state decreased steeply at the shear stress level of 2.5 $dyne/cm^2$. And the cell death rate also showed dramatic decrease of about 33.7 % at the shear stress level of 2.5 $dyne/cm^2$. These results suggests that the cellular response to toxic decreases at the shear stress level of $dyne/cm^2$ and impedance analysis is capable of analyzing the fine toxic response hard to characterize with the visual detachment. In the thesis, we have designed, fabricated, and characterized two cell adhesion chips (cell adhesion chip based on visual detachment observation and cell adhesion chip based on visual detachment observation and impedance analysis). The present chips have potential for use in cancer metastasis study and drug response analysis through the cell adhesion analysis.