2.3. DEP Characterization of ES-B3 and MEF Cells

To sort ES-B3 and MEF cells from the mixed cell suspension, the DEP characteristics of ES-B3 and MEF cells were evaluated. To determine the crossover frequency between negative- and positive-DEP, the behavior of each cell was evaluated under various AC voltage frequencies. To cause the DEP phenomenon, a non-equal electric field was generated using transparent conductive glass (Figure 1) [37,39]. This chamber consisted of a transparent parallel-line electrode array on a glass substrate, ITO-coated glass, and a silicone rubber gasket. The parallel-line electrode array was fabricated using ITO-coated glass (Geomatec Co., Ltd., Yokohama, Japan) as a conductive substrate. The thickness of the ITO layer was 1500 Å, and the resistance was 5 Ω/sq. The parallel-line electrode was patterned using laser etching techniques. The electrode array was designed to generate a highly non-uniform electric field [37,39]. The width of each electrode line was 20 μm, and the spaces between each electrode were 80 μm (Figure 1a). The flow channel was made from a silicon rubber gasket to make a rectangular volume. The DEP chamber was formed by sandwiching the silicon rubber gasket between the parallel-line electrode array and a bare ITO-coated slide glass drilled with holes for the fluidic inlet and outlet. The thickness of the silicon rubber gasket was 500 μm. The cells were moved toward the electrodes by p-DEP and between electrodes by n-DEP in the DEP chamber (Figure 1b). The AC electric field was applied between the parallel-line electrode array and bare ITO-coated glass, using a function generator (WF1974, NF Corp., Yokohama, Japan) and amplifier (BA4850, NF Corp., Yokohama, Japan). The applied voltage was monitored by an oscilloscope (TDS1001B, Tektronix, Beaverton, OR, USA) connected in parallel. The movements of the cells within the DEP chamber were observed using a phase-contrast microscope (Nikon Eclipse TE300, Nikon, Tokyo, Japan) with a digital video camera.

Dielectrophoretic characterization of living cells: (a) Schematic of dielectrophoresis (DEP) chamber; (b) Positive- and negative- DEP of living cells; (c) Discrimination of positive- and negative-DEP.

For the DEP characterization, ES-B3 or MEF cell suspension in LCB was injected and subjected to an AC electric field 180 s after injection. The magnitude of the imposed AC voltage was 20 V_p-p, and the frequency was varied from 10 kHz to 1 MHz. The behavior of ES-B3 and MEF cells was observed by the digital camera on the microscope, and microphotographs were captured 180 s after each AC voltage frequency was imposed. The captured images were trimmed to 200 μm × 300 μm, and the number of cells on the electrodes (positive-DEP) and between the electrodes (negative-DEP) were counted. The ratio of cells indicating positive-DEP in the chamber was calculated to evaluate the crossover frequency. The dielectrophoretic property of a cell (indicating p-DEP or n-DEP) was assessed based on the region where the cell moved (Figure 1c). The frequency dependency of the DEP property was also evaluated as:

where N_P and N_N are the number of cells indicating positive-DEP and negative-DEP, respectively. When the ratio of cells indicating positive-DEP is 50%, the frequency of the AC voltage is considered a transition point from negative-DEP to positive-DEP. In this study, the crossover frequency of the DEP is defined as the frequency at which the ratio of cells indicating positive-DEP is 50%.