Correlation of the exposed-cell mortality with the transient cavitation noise in vitro

The cavitation noise is a suitable and accurate indicator of the cavitation activity induced in a liquid. Frohly (2000) proposed a quantitative indicator of the transient cavitation calculated as a cavitation spectrum integration in a logarithmic scale and called CNP cavitation noise power indicator. This work studied the relation between a new indicator CMS based on CNP and the exposed-cell mortality in the following conditions. The cell suspension to be insonified (prostatic cells AT2 at 2.5 10⁶ cells/mL) is placed in a medium sample tray (12 wells/tray, 2 mL/well, well diameter: 20 mm). This tray is submerged at mid-depth in degassed water and positioned 5 mm above the face of a flat ultrasonic transducer (diameter 22 mm, frequency: 445.5 kHz; intensity: 0.08-1.09 W/cm², exposure time: 30 sec-4 min). This technical configuration was admitted to be conducive to standing-wave generation through reflection at the air/medium interface in the well thus enhancing the cavitation phenomenon. Laterally to the transducer, a homemade hydrophone (PVDF film of 10 min diameter molded in araldite AY103) was oriented to receive the acoustical signal from the bubbles. From this spectral signal (0.1 to 7.1 MHz bandwidth) recorded every 3 sec on a computer during the exposure condition, CNP was calculated. Its mean value was compared to the cell mortality measured just after ultrasound exposure with a flow cytometer (FACScan) by counting 10000 events. This was accomplished by adding 7AAD solution (Via-Probe kit). Ten exposure conditions were chosen. Two of them corresponded to the experimental limits: no effects on cells (i.e. 0.08 W/cm²; 4 min) - complete destruction of cells (i.e. 1.09 W/cm²; 30 sec). 3 to 8 measures were realized for each exposure condition. The mortality due to cavitation effect varied from 0.1% to 85.5% and was compared to CMS mean given in relative value (from 3.8% to 97.5%). A simply linear relation between these parameters was given by a correlation coefficient of r²=0.81. This correlation coefficient came better when exposed times were considered (for 1 min r²=0.82 and for 2 min r²=0.95). These results show that CNP is a good indicator of the effect induced by the cavitation phenomeno- n in cell culture medium in vitro.