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.