Review of Recent Results of Onco-Ultrasound-Tripsy on Cancer Cells 5 Cells were exposed for 180 seconds at a fixed frequency in pulse mode, with a burst rate of 10 Hz, a 50% duty cycle, and 60 V voltage. Initially, different frequencies were tested to identify those that caused the lowest percentage of live cancer cells post-sonication. Five emission frequencies were evaluated: 400, 510, 582, 800, and 1000 kHz, with a power density always below 5.4 W/cm2. After these preliminary tests, two frequencies (800 and 1000 kHz) were found to result in the lowest percentage of surviving cells post-sonication. These two frequencies were then tested again on two separate days, with three replicates of the same experimental conditions for each day. Additionally, the cell growth index was monitored in real time following ultrasound treatment. This was achieved by using a device that measures changes in cellular impedance, where an increase in the number of adherent cells corresponds to a rise in impedance, serving as an indicator of cell growth. These growth indices were then compared to that of untreated cells. The experiments were conducted using the xCELLigence RTCA instrument (ACEA Biosciences, Inc., CA, USA), in a humidified incubator maintained at 37◦C with 95% air and 5%CO2. Figure 3 (Left) presents the percentage of dead cells following sonication for five different tested frequencies: 400, 510, 582, 800, and 1000 kHz. The evaluation of dead cells was carried out using Trypan blue assays, as dead cells are permeable to the dye and can thus be counted. This procedure was performed immediately after the sonication process, and the number of dead cells was compared with the initial number of treated cells to calculate the percentage shown in Figure 3. The data reveals a general increase in the percentage of dead cells as the frequency rises, particularly for frequencies above 600 kHz. The results at 400 kHz should be considered an outlier and warrant further investigation. The highest cancer cell mortality was observed at the two highest frequencies: 45% of cells were dead after sonication at 800 kHz, and 60% of cells were dead at 1000 kHz. Fig. 3 (Left) Percentage of dead cells after sonication at the five selected frequencies; (Middle) follow-up of the live cells percentage two days after the sonication; (Right) Recalculation of the live cells percentage including considerations about cell/fragment diameter. The experimental results from the following two days of testing for the selected frequencies of 800 and 1000 kHz are presented in Figure 3 (Middle). The sonication experiments confirm that 1000 kHz is the frequency that results in the lowest percentage of live cancer cells post-sonication, with an average value of under 36%. Further evaluation was conducted to investigate the possibility that live cells might be present in the culture medium after treatment, potentially detached from the bottom of the dish by the ongoing pressure wave but not damaged by the ultrasound. To rule out this hypothesis, which could result in an overestimation of the number of killed cells, the culture medium was centrifuged to collect the pellet. The pellet was then cultured, and the corresponding growth index was assessed. For all the analyzed dishes, the growth index was found to be zero, indicating the absence of live cells in the medium. Results of this new calculation are presented in Fig. 3 (Right) where analysis is restricted only to cells in the range between 12 and 25µm. Application on Melanoma Cells In order to assess the effects of low-intensity ultrasound on melanoma cells, the HBL metastatic cell line was utilized, known for its high proliferative capacity. These cells were derived from human biopsies at the Department of Translational Biomedicine and Neuroscience (DiBraiN) at the University of Bari “Aldo Moro.” Cells were cultured in Dulbecco’s Modified Eagle’s Medium High Glucose (DMEM H.G.), supplemented with 10% fetal bovine serum (FBS), 1% L-Glutamine (Euroclone), and 1% Penicillin/Streptomycin (Full DMEM/HG). All cultures were maintained in an incubator at 37◦C with 95% humidity and 5% CO2.
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