Department of Biomedical Engineering, The George Washington University, Washington, DC, USA.
Department of Biomedical Engineering, The George Washington University, Washington, DC, USA.
Ultrasound Med Biol. 2022 Jun;48(6):1078-1094. doi: 10.1016/j.ultrasmedbio.2022.02.006. Epub 2022 Mar 15.
To the best of our knowledge, therapeutic ultrasound (TUS) is thus far an unexplored means of delivering mechanical stimulation to cardiomyocyte cultures, which is necessary to engineer a more mature cardiomyocyte phenotype in vitro. Spectral ultrasound (SUS) may provide a way to non-invasively, non-disruptively and inexpensively monitor growth and change in cell cultures over long periods. Compared with other measurement methods, SUS as an acoustic measurement tool will not be affected by an acoustic therapy, unlike electrical measurement methods, in which motion caused by acoustic therapy can affect measurements. Further SUS has the potential to provide functional as well as morphological information in cell cultures. Human induced pluripotent stem cell cardiomyocytes (iPS-CMs) were imaged with calcium fluorescence microscopy while TUS was being applied. TUS was applied at 600 kHz and 1, 3.4 and 6 W/cm for a continuous 1 s pulse. Measures of the instantaneous beat frequency, repolarization rate and calcium spike amplitude were calculated from the fluorescence data. At 600 kHz, TUS at 1 and 6 W/cm had significant effects on the shortening of both the repolarization rate and instantaneous beat rate of the iPS-CMs (p < 0.05), while TUS at 3.4 and 6 W/cm had significant effects on the shortening of the calcium spike amplitude (p < 0.05). Three SUS measures and one gray-level measure were captured from the iPS-CM monolayers while they were simultaneously being imaged with calcium-labeled confocal microscopy. The gray-level measure performed the best of all SUS measures; however, it was not reliable enough to produce a consistent determination of the beat rate of the cell. Finally, SUS measures were captured using three different transducers while simultaneously applying TUS. A center-of-mass (COM) measure calculated from the wavelet transform scalogram of the time-averaged radiofrequency data revealed that SUS was able to detect a change in the frequency content of the reflected ultrasound at 1 and 6 W/cm before and after ultrasound application (p < 0.05), showing promise for the ability of SUS to measure changes in the beating behavior of iPS-CMs. Overall, SUS is promising as a method for constant monitoring of dynamic cell and tissue culture and growth.
据我们所知，治疗超声（TUS）迄今为止尚未被探索用于向心肌细胞培养物传递机械刺激，而这对于在体外构建更成熟的心肌细胞表型是必要的。光谱超声（SUS）可能提供一种非侵入性、非破坏性且廉价的方法来长期监测细胞培养物的生长和变化。与其他测量方法相比，SUS 作为声学测量工具，不会像电测量方法那样受到声疗的影响，因为声疗引起的运动可能会影响测量。此外，SUS 有可能在细胞培养物中提供功能和形态信息。在应用 TUS 的同时，使用钙荧光显微镜对人诱导多能干细胞心肌细胞（iPS-CMs）进行成像。TUS 以 600 kHz 及 1、3.4 和 6 W/cm 的连续 1 s 脉冲施加。从荧光数据中计算瞬时跳动频率、复极化率和钙峰幅度的测量值。在 600 kHz 时，TUS 在 1 和 6 W/cm 对 iPS-CMs 的复极化率和瞬时跳动率缩短均有显著影响（p < 0.05），而 TUS 在 3.4 和 6 W/cm 对钙峰幅度缩短有显著影响（p < 0.05）。在同时用钙标记共聚焦显微镜对 iPS-CM 单层成像的同时，从 iPS-CM 单层中捕获了三个 SUS 测量值和一个灰度测量值。灰度测量值在所有 SUS 测量值中表现最好；然而，它不够可靠，无法对细胞跳动率进行一致的确定。最后，同时应用 TUS 时，使用三个不同的换能器捕获 SUS 测量值。从时均射频数据的小波变换谱图计算出的质心（COM）测量值表明，SUS 能够在应用超声前后检测到 1 和 6 W/cm 反射超声的频率内容变化（p < 0.05），这表明 SUS 有能力测量 iPS-CMs 跳动行为的变化。总体而言，SUS 有望成为一种用于持续监测动态细胞和组织培养物及生长的方法。
