4 C. Pappalettere and G. Pappalettera minutes. Then, 1.75 mL of medium was added, mixed, followed by the addition of the previous supernatant, PBS, and trypan blue and finally centrifugate at 1200 rpm for 3 mins. The following sonication conditions were chosen: a sinusoidal wave in pulsed wave mode, with a pulse repetition frequency of 10 Hz, a 50% duty cycle, and 7 fixed frequencies of 400 kHz, 436 kHz, 472 kHz, 510 kHz, 546 kHz, 582 kHz, and 620 kHz. The tests were conducted either at a constant voltage of 60 V or at a constant power of 0.1 W. This power value represents the average output power across the frequency range of 400–620 kHz and also the median power among the 7 sonication frequencies. Results in terms of observed mortality are shown in Fig. 2. Fig. 2 Response in terms of cell mortality for the fixed voltage condition (green square) and the fixed power condition (red circles) Figure 2 shows the trend of cell mortality while varying the frequency. The two tested conditions show quite similar trends indicating that cell mortality is mostly correlated with frequency. More specifically cell mortality rises 10% to 15% at 400 kHz to 55% to 60% at 620 kHz. However, this increase is concentrated in the 540–620 kHz frequency range. Application on Osteosarcoma Cells The MG-63 osteosarcoma (OS) cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) [25]. Cells were seeded at a density of 8×104 cells per T25 flask and incubated at 37◦Cin a standard environment with 95% air and 5% CO2. To detach the cells, Trypsin/EDTA was used, after which the cells were re-plated in Petri dishes and returned to the incubator. Cell confluence was monitored using a Nikon Eclipse Ti-U inverted microscope with a C1 Digital Eclipse modular confocal system. Cells were exposed for 180 s at a fixed frequency in pulse mode, with a burst rate of 10 Hz, 50% duty cycle and 60 V voltage. In an initial phase, different frequencies were tested to identify the ones able to induce the lowest % of live cancer cells after sonication. Five emission frequencies were analyzed: 400, 510, 582, 800 and 1000 kHz. The power density was, in any case, lower than 5.4 W/cm2. After these preliminary tests, two frequencies were found associated with the lower percentage of living cells after sonication: 800 and 1000 kHz. The sonication tests for these two frequencies were then repeated on two different days with three replications of the same experimental conditions for each day. Moreover, we the growth index of the cells after ultrasound treatment in real time was analyzed. This was done by using a device able to measure variation in cellular impedance. In fact, an increment of the number of adherent cells results in an increment of the measured impedance and this can be used as an indicator of the cellular growth. These growth indexes were then compared with the growth index of the untreated cells. Experiments were carried out using the xCELLigence RTCA instrument (ACEA Biosciences, Inc., CA, USA), placed in a humidified incubator maintained at 37◦C with 95% air and 5% CO2.
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