Materials & Methods on Cell Experiments

Cell culture

U251 glioma cells was purchased from Qingqi Biotechnology Development Co., Ltd. (Shanghai, China). U251 cells were cultured in RPMI-1640 complete medium (containing 100 U/mL penicillin and 100 U/mL streptomycin, 10% inactivated fetal bovine serum) at 37℃ under 5% CO2 in the culture dish. The cell suspension was prepared with 2 mL of 0.25% trypsin-EDTA when 80% confluency was reached. After two minutes, 2 mL of 1640 medium was added to inactivate trypsin-EDTA and centrifuged at 800 rpm for 5 minutes at room temperature. Then, the supernatant was discarded, and the cell culture was re-suspended, configuring to a cell suspension with 1×105 cells/mL. The cell suspension was were seeded into each well of six-well plates with 1 mL. After twelve hours, the cells were treated by high frequency AC field.

Experimental setups

The insulated electrode sheet (Fig. 6a) was designed to deliver high frequency AC field. The insulated electrode consists of a PCB board, insulating ceramic sheet, and silica gel, where the PCB board was designed to provide support and connection between electrode and generator. The top pad of the PCB board connected to the power output, while the bottom pad was linked to the ceramic sheet via conductive silver paste, with silica gel filling the gap to prevent direct contact between the conductive parts and the cell culture medium. The PCB board measures 25.24 mm in length, 1.6 mm in width, and 19 mm in height. The distance between the two electrode sheets in the six-well cell culture plate is 20.18 mm. The dimensions of the ceramic sheet are 19.05 mm × 9.53 mm × 0.2 mm. A PCB holder was designed to secure six electrode plates in the six-well cell culture plate, allowing three sets of experiments to be conducted simultaneously. Thin wires connected the power supply to the PCB holder, ensuring the six-well cell plate remained sealed, thus providing an uncontaminated environment for the cells (Fig. 6b). The experimental setup is shown in Fig. 6c.