In vitro ultrasound experiments: Standing wave and multiple reflections influence on the outcome

The purpose of this work was to determine the influence of standing waves and possible multiple reflections under the conditions often encountered in examining the effects of ultrasound exposure on the cell cultures in vitro. More specifically, the goal was to quantitatively ascertain the influence of ultrasound exposure under free field (FF) and standing waves (SW) and multiple reflections (MR) conditions (SWMR) on the biological endpoint (50% cell necrosis). Such information would help in designing the experiments, in which the geometry of the container with biological tissue may prevent FF conditions to be established and in which the ultrasound generated temperature elevation is undesirable. This goal was accomplished by performing systematic, side-by-side experiments in vitro with C6 rat glioma cancer cells using 12 well and 96 well plates. It was determined that to obtain 50% of cell viability using the 12 well plates, the spatial average, temporal average (I_SATA) intensities of 0.32W/cm² and 5.89W/cm² were needed under SWMR and FF conditions, respectively. For 96 well plates the results were 0.80W/cm² and 2.86W/cm² respectively. The corresponding, hydrophone measured p_RMS maximum pressure amplitude values, were 0.71MPa, 0.75MPa, 0.75MPa and 0.73MPa, respectively. These results suggest that p_RMS pressure amplitude was independent of the measurement set-up geometry and hence could be used to predict the cells' mortality threshold under any in vitro experimental conditions or even as a starting point for (pre-clinical) in vivo tests. The described procedure of the hydrophone measurements of the p_RMS maximum pressure amplitude at the λ/2 distance (here 0.75mm) from the cell's level at the bottom of the dish or plate provides the guideline allowing the difference between the FF and SWMR conditions to be determined in any experimental setup. The outcome of the measurements also indicates that SWMR exposure might be useful at any ultrasound assisted therapy experiments as it permits to reduce thermal effects. Although the results presented are valid for the experimental conditions used in this study they can be generalized. The analysis developed provides methodology facilitating independent laboratories to determine their specific ultrasound exposure parameters for a given biological end-point under standing waves and multiple reflections conditions. The analysis also permits verification of the outcome of the experiments mimicking pre- and clinical environment between different, unaffiliated teams of researchers.