Introduction Electrical properties of cells (i.e. membrane capacitance and cytoplasm conductivity) have been studied to understand the complex physiological states of cells and used as markers for determining cell types. Electrorotation (ROT) that is one of the alternating current electrokinetic phenomena has been utilized to characterize the electrical properties of single cells. In ROT techniques, quadrupole electrodes were conventionally employed to induce a rotating electric field at the center of them by applying sine waves with 90° differences of each phase. However, a relatively long experimental period was required because several single cells must be repeatedly arranged to a center of quadrupole electrodes.In this presentation, we demonstrated the simultaneous ROT measurement of K562 cells by using the 3D-interdigitated array (3D-IDA) device to determine the membrane capacitances and cytoplasm conductivities of the different types of cells [1]. The rotating electric fields in over 1,500 grids were simultaneously generated by this device to obtain the distribution of rotation rates of over 50 cells with single operation [2]. The grid is defined as regions surrounded by four microband electrodes. We have also developed a new 3D-IDA device with microwells to maintain the cells in them during a chemical stimulation by a fluid flow. We investigated ROT rates accompanied with the changes of the dielectric properties of cells during the stimulation with an activator regent for Jurkat cells. This is the first report to monitor the changes of ROT rates upon chemical stimuli to investigate the effect of chemical agents to cellular function. Experimental Methods Construction of 3D-IDA device The device consisted of two IDA electrodes (20 μm in width, 30 μm gaps) made from indium tin-oxide (ITO). The IDA electrode was overlaid on another IDA electrode by an orthogonal arrangement via a double-sided adhesive film (30 μm in thickness) (Fig. A). A constant rotational electric field was generated in the grids by applying AC voltages with 90° differences of each phase to four microband electrodes. Construction of 3D-IDA device with microwells Microwells with rectangle shape (26 mm in long side and 20 mm in short side) were fabricated with an insulative photoresist (20 mm in thickness) on the gaps of ITO-IDA electrodes. Two ITO-electrodes (3 µm × 20 µm) were arranged at both short sides on the bottoms of microwells. Two gold electrodes (3 µm × 20 µm) were also arranged at both long sides on the insulative photoresist (Fig. B). Results and Conclusions Simultaneous ROT measurement of K562 cells using the 3D-IDA device K562 cells were resuspended in a ROT solution with the conductivity of 360 mS m−1 (the mixture of 75% (v/v) of 300 mM mannitol and 25% (v/v) of RPMI 1640 medium) and subjected to ROT measurement by 3D-IDA devices. When AC signals (700 kHz, 5 Vpp) were applied, K562 cells dispersed randomly started to rotate and move to the center of each grid. The ratio of the grids occupied with single cells was 45%, when the cell suspension with the concentration of 4.0 × 107 cells mL-1 was injected (Fig, A). Thus, we could obtain the rotation rates of 450–680 cells (40–60 cells in single images) simultaneously and estimated the ROT rate as 8.53 radian s-1(Fig. C). Monitoring of ROT behavior of single cells stimulated with calcium ionophore The ROT device with the microwells were filled with Jurkat cells resuspended in the ROT solution adjusted to 5 × 105 cells mL-1 and incubated for 2.5 min to arrange cells in the microwells. The ratio of microwells occupied with single cells were 27% (Fig. B). When AC voltages (2 Vpp, 300 kHz) with different phases were applied to four electrodes, the cells began to rotate (Fig. D). The average ROT rate of Jurkat cells was estimated and found to be 1.01 radian s-1. Then, the solution containing 1 mM ionomycin which is well known as an activator for Jurkat cells to lead to the decrease of the membrane capacitance [3] was injected in the device. Although the solution above the microwells was disturbed by the fluidic flow (40 mm s-1), the cells rotated with the constant rate in the microwells. The ROT rate gradually decreased and reached at 0.73 radian s-1 40 s after the injection. The result could reflect the decrease of the membrane capacitance due to the activation of Jurkat cells induced by the stimulus of ionomycin. The 3D-IDA device with microwells would be applied to the high through-put screening of drugs and discrimination of highly responsible cells to drugs. References (1) Ino, K.; Ishida, A.; Inoue, K. Y.; Suzuki, M.; Koide, M.; Yasukawa, T.; Shiku, H.; Matsue, T. Electrorotation Chip Consisting of Three-Dimensional Interdigitated Array Electrodes. Sensors Actuators, B Chem. 2011, 153 (2), 468–473. https://doi.org/10.1016/j.snb.2010.11.012.(2) Kawai, S.; Suzuki, M.; Arimoto, S.; Korenaga, T.; Yasukawa, T. Determination of Membrane Capacitance and Cytoplasm Conductivity by Simultaneous Electrorotation. Analyst 2020, 145 (12), 4188–4195. https://doi.org/10.1039/d0an00100g.(3) Pethig, R.; Bressler, V.; Carswell‐Crumpton, C.; Chen, Y.; Foster‐Haje, L.; García‐Ojeda, M. E.; Lee, R. S.; Lock, G. M.; Talary, M. S.; Tate, K. M. Dielectrophoretic Studies of the Activation of Human T Lymphocytes Using a Newly Developed Cell Profiling System. Electrophoresis 2002, 23 (13), 2057–2063. https://doi.org/10.1002/1522-2683(200207)23:13<2057::AID-ELPS2057>3.0.CO;2-X. Figure 1
Search from 250 M+ research papersSearch from 250 M+ research papers
Sep 28, 2023
Adi Haber + 4
Open Access