## 4 Discussion
### Cell Viability after LIPUS with Different Setups  
Diverse experimental setups for in vitro cell exposure with LIPUS have been described in the literature with the support of models and hydrophone measurements [12]. Due to their low cost, practicality and application to adherent cells, we evaluated two experimental setups: the well-on-transducer and the well-on-water-surface configurations. We observed that LIPUS applied with the gel-based setup presents greater detrimental effects on cell viability than LIPUS application with the water-based setup at similar acoustic conditions (Figure 2a,b). Although the acoustic energy applied in both cases was the same, the viability of cells treated with LIPUS for the first case decreased at acoustic intensities larger than 0.4 W/cm\({}^{2}\) at 50% DF (\(p<0.001\)), while the cells treated using the well-on-water-surface setup tolerated larger intensities with a significant viability decrease only at 1.6 W/cm\({}^{2}\) at 50% DF (\(p<0.01\)). Similar results were observed in the viability decline of the hepatocarcinoma cell line, SMMC-7721, at 1 min stimulation with 0.5 W/cm\({}^{2}\) (0.25% DF) or higher LIPUS intensities using the well-on-transducer setup [1]. In contrast, HepG2 and 3T3 cells exhibited significant viability reductions only at high LIPUS intensities of 1.2 W/cm\({}^{2}\) at 50% DF when stimulated in a well-on-water-surface configuration [10]. According to [12], both studied setups present the disadvantage that variations in the height of the culture medium, which determines the distance between the cell monolayer and the air interface, impact the amplitude of acoustic pressure. Nevertheless, the well-on-water-surface setup tolerates larger variations in media volume than the well-on-transducer configuration; for instance, the former presents a 10% reduction in pressure amplitude when the height deviates 0.46 mm, while the latter displays the same reduction for variations of 0.02 mm in media's height. This particularity of the well-on-water-surface setup should permit to have more robust results among repetitions. Figure 4: 2D axisymmetric modeled temperature for the experiments with ultrasound at 1.6 W/cm\({}^{2}\) at 50% DF after 5 min. A maximum temperature increment of 0.76 \({}^{\circ}\)C should not affect cell viability. This figure represents the result with the most intense experimental condition used in this work. Agitation produced by the acoustic force, not considered here, would also uniformize the heat in experiments, which would reduce the average temperature. However, two additional hypotheses could complement the explanation of the differences in the cell viability between gel-based or water-based setups, which are not mutually excluding. First, when using the well-on-transducer setup, an extra mechanical vibration from the transducer could be transmitted by direct contact to the culture dish. This additional vibrational wave may increase the overall cellular effect when combined with the pure ultrasound propagation; this combination of phenomena was already proposed to be a significant contributor for the whole ultrasonic effects in other applications [29]. Second, the resonance effects may be increased when the transducer is directly coupled to the dish, probably due to the parallelism of the multiple interfaces that enhance the possibility of producing standing waves in media [12]. Both, mechanical vibration and resonance effects may be the leading factors that cause the instant cell detachment observed with the ultrasound stimulation at 0.8 to 1.6 W/cm\({}^{2}\) and 50% DF (data not shown). In contrast, as both phenomena are reduced in the well-on-water-surface setup, there is a marked reduction in the instantaneous cell lysis, but instead, cells undergo delayed cell death processes which comprise early and secondary apoptosis [1,8].