Secomski Wojciech, Bilmin Krzysztof, Kujawska Tamara, Nowicki Andrzej, Grieb Paweł, Lewin Peter A
Institute of Fundamental Technological Research, Polish Academy of Sciences, Pawińskiego 5b, 02-106 Warszawa, Poland.
Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego 5, 02-106 Warszawa, Poland.
Ultrasonics. 2017 May;77:203-213. doi: 10.1016/j.ultras.2017.02.008. Epub 2017 Feb 20.
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) intensities of 0.32W/cm and 5.89W/cm were needed under SWMR and FF conditions, respectively. For 96 well plates the results were 0.80W/cm and 2.86W/cm respectively. The corresponding, hydrophone measured p maximum pressure amplitude values, were 0.71MPa, 0.75MPa, 0.75MPa and 0.73MPa, respectively. These results suggest that p 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 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.
这项工作的目的是确定在体外检测超声暴露对细胞培养物影响时经常遇到的条件下驻波和可能的多次反射的影响。更具体地说，目标是定量确定在自由场（FF）、驻波（SW）和多次反射（MR）条件（SWMR）下超声暴露对生物学终点（50%细胞坏死）的影响。此类信息将有助于设计实验，在这些实验中，装有生物组织的容器的几何形状可能会阻止建立FF条件，并且超声产生的温度升高是不可取的。通过使用12孔板和96孔板对C6大鼠胶质瘤癌细胞进行系统的体外并行实验实现了这一目标。已确定，使用12孔板要获得50%的细胞活力，在SWMR和FF条件下分别需要0.32W/cm²和5.89W/cm²的空间平均、时间平均（I）强度。对于96孔板，结果分别为0.80W/cm²和2.86W/cm²。相应的水听器测量的p最大压力幅值分别为0.71MPa、0.75MPa、0.75MPa和0.73MPa。这些结果表明，p压力幅值与测量设置的几何形状无关，因此可用于预测任何体外实验条件下细胞的死亡阈值，甚至可作为（临床前）体内试验的起点。所描述的在距培养皿或平板底部细胞水平λ/2距离（此处为0.75mm）处测量p最大压力幅值的水听器测量程序提供了指导方针，可用于在任何实验设置中确定FF和SWMR条件之间的差异。测量结果还表明，SWMR暴露在任何超声辅助治疗实验中可能都有用，因为它可以减少热效应。尽管所呈现的结果对于本研究中使用的实验条件是有效的，但它们可以推广。所开展的分析提供了一种方法，便于独立实验室在驻波和多次反射条件下为给定的生物学终点确定其特定超声暴露参数。该分析还允许验证不同、无关联的研究团队之间模拟临床前和临床环境的实验结果。
