Therapeutic Ultrasound Effects on Human iPS-CM Cells Measured Optically and with Spectral Ultrasound

• Published in: Ultrasound in medicine & biology Vol. 48; no. 6; pp. 1078 - 1094
• Main Authors: Chen, Andrew W., Saab, George, Jeremic, Aleksandar, Zderic, Vesna
• Format: Journal Article
• Language: English
• Published: 15.03.2022
• ISSN: 0301-5629; 1879-291X
• DOI: 10.1016/j.ultrasmedbio.2022.02.006

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 of time. Compared to other measurement methods, SUS as an acoustic measurement tool will not be affected by an acoustic therapy, unlike electrical measurement methods where motion caused by acoustic therapy can impact our 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 2 for a continuous one second pulse. Measures of the instantaneous beat frequency, repolarization rate and calcium spike amplitude were calculated from the fluorescence data. 600 kHz TUS at 600 kHz 1 W/cm 2 and 6 W/cm 2 had significant effects in the shortening of both the repolarization rate as well as the instantaneous beat rate of the iPS-CMs (p<0.05) while TUS at 3.4 W/cm 2 and 6 W/cm 2 had significant effects in 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 out 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 RF data revealed that SUS was able to detect a change in the frequency content of the reflected ultrasound at 1 and 6 W/cm 2 before and after ultrasound application (p<0.05) showing promise for SUS to measure changes in the beating behavior of the iPS-CMs. Overall, SUS shows promise as a method for constant monitoring of dynamic cell and tissue culture and growth.