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Ultrasound field characterization and bioeffects in multiwell culture plates.

Patel US, Ghorayeb SR, Yamashita Y, Atanda F, Walmsley AD, Scheven BA - J Ther Ultrasound (2015)

Bottom Line: Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer.The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells.Significant thermal variations were not detected in adjacent untreated wells.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St Chad's Queensway, Birmingham, B4 6NN UK.

ABSTRACT

Background: Ultrasound with frequencies in the kilohertz range has been demonstrated to promote biological effects and has been suggested as a non-invasive tool for tissue healing and repair. However, many challenges exist to characterize and develop kilohertz ultrasound for therapy. In particular there is a limited evidence-based guidance and standard procedure in the literature concerning the methodology of exposing biological cells to ultrasound in vitro.

Methods: This study characterized a 45-kHz low-frequency ultrasound at three different preset intensity levels (10, 25, and 75 mW/cm(2)) and compared this with the thermal and biological effects seen in a 6-well culture setup using murine odontoblast-like cells (MDPC-23). Ultrasound was produced from a commercially available ultrasound-therapy system, and measurements were recorded using a needle hydrophone in a water tank. The transducer was displaced horizontally and vertically from the hydrophone to plot the lateral spread of ultrasound energy. Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer. During ultrasound treatment, cell cultures were directly exposed to ultrasound by submerging the ultrasound transducer into the culture media. Four groups of cell culture samples were treated with ultrasound. Three with ultrasound at an intensity level of 10, 25, and 75 mW/cm(2), respectively, and the final group underwent a sham treatment with no ultrasound. Cell proliferation and viability were analyzed from each group 8 days after three ultrasound treatments, each separated by 48 h.

Results: The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells. Significant thermal variations were not detected in adjacent untreated wells.

Conclusions: This study highlights the pitfalls of using multiwell plates when investigating the biological effect of kilohertz low-frequency ultrasound on adherent cell cultures.

No MeSH data available.


Temperature changes with ultrasound treatment. Temperature changes in culture medium in a well of the six-well plate which is directly exposed with 45-kHz ultrasound over a 30-min period
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Fig5: Temperature changes with ultrasound treatment. Temperature changes in culture medium in a well of the six-well plate which is directly exposed with 45-kHz ultrasound over a 30-min period

Mentions: Temperature measurements indicated that ultrasound with a frequency of 45 kHz, and at the three specified intensities, did not significantly affect the temperature of the culture medium in culture wells adjacent to and distant from the well being treated with ultrasound. Measurements also confirmed that the water bath setup was able to keep the temperature of the culture medium stable at 37 °C (±1 °C). Figure 5 shows the temperature rise in the culture medium of the culture well with the transducer submerged and producing ultrasound. The highest of the three intensities, 75 mW/cm2, produced a temperature rise of nearly 16 °C after 30 min of ultrasound exposure. Intensities of 10 and 25 mW/cm2 increased the temperature of the medium resulting in maximum temperatures of 4 and 7 °C, respectively, over 30 min of ultrasound exposure. It was observed for the lower two intensities, the temperature rise reached a plateau before the maximum treatment time of the device was reached. This did not occur at the highest intensity. After 5 min (300 sec) of ultrasound treatment, the temperature of the culture medium had risen by 1.6, 3, and 5.5 °C with intensities, 10, 25, and 75 mW/cm2, respectively. These data indicate that treatment with ultrasound of a short duration using this method only marginally increases the ambient temperature of the culture medium, but longer times up to 30 min can generate a significant temperature rise.Fig. 5


Ultrasound field characterization and bioeffects in multiwell culture plates.

Patel US, Ghorayeb SR, Yamashita Y, Atanda F, Walmsley AD, Scheven BA - J Ther Ultrasound (2015)

Temperature changes with ultrasound treatment. Temperature changes in culture medium in a well of the six-well plate which is directly exposed with 45-kHz ultrasound over a 30-min period
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4490766&req=5

Fig5: Temperature changes with ultrasound treatment. Temperature changes in culture medium in a well of the six-well plate which is directly exposed with 45-kHz ultrasound over a 30-min period
Mentions: Temperature measurements indicated that ultrasound with a frequency of 45 kHz, and at the three specified intensities, did not significantly affect the temperature of the culture medium in culture wells adjacent to and distant from the well being treated with ultrasound. Measurements also confirmed that the water bath setup was able to keep the temperature of the culture medium stable at 37 °C (±1 °C). Figure 5 shows the temperature rise in the culture medium of the culture well with the transducer submerged and producing ultrasound. The highest of the three intensities, 75 mW/cm2, produced a temperature rise of nearly 16 °C after 30 min of ultrasound exposure. Intensities of 10 and 25 mW/cm2 increased the temperature of the medium resulting in maximum temperatures of 4 and 7 °C, respectively, over 30 min of ultrasound exposure. It was observed for the lower two intensities, the temperature rise reached a plateau before the maximum treatment time of the device was reached. This did not occur at the highest intensity. After 5 min (300 sec) of ultrasound treatment, the temperature of the culture medium had risen by 1.6, 3, and 5.5 °C with intensities, 10, 25, and 75 mW/cm2, respectively. These data indicate that treatment with ultrasound of a short duration using this method only marginally increases the ambient temperature of the culture medium, but longer times up to 30 min can generate a significant temperature rise.Fig. 5

Bottom Line: Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer.The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells.Significant thermal variations were not detected in adjacent untreated wells.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St Chad's Queensway, Birmingham, B4 6NN UK.

ABSTRACT

Background: Ultrasound with frequencies in the kilohertz range has been demonstrated to promote biological effects and has been suggested as a non-invasive tool for tissue healing and repair. However, many challenges exist to characterize and develop kilohertz ultrasound for therapy. In particular there is a limited evidence-based guidance and standard procedure in the literature concerning the methodology of exposing biological cells to ultrasound in vitro.

Methods: This study characterized a 45-kHz low-frequency ultrasound at three different preset intensity levels (10, 25, and 75 mW/cm(2)) and compared this with the thermal and biological effects seen in a 6-well culture setup using murine odontoblast-like cells (MDPC-23). Ultrasound was produced from a commercially available ultrasound-therapy system, and measurements were recorded using a needle hydrophone in a water tank. The transducer was displaced horizontally and vertically from the hydrophone to plot the lateral spread of ultrasound energy. Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer. During ultrasound treatment, cell cultures were directly exposed to ultrasound by submerging the ultrasound transducer into the culture media. Four groups of cell culture samples were treated with ultrasound. Three with ultrasound at an intensity level of 10, 25, and 75 mW/cm(2), respectively, and the final group underwent a sham treatment with no ultrasound. Cell proliferation and viability were analyzed from each group 8 days after three ultrasound treatments, each separated by 48 h.

Results: The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells. Significant thermal variations were not detected in adjacent untreated wells.

Conclusions: This study highlights the pitfalls of using multiwell plates when investigating the biological effect of kilohertz low-frequency ultrasound on adherent cell cultures.

No MeSH data available.